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Ecotoxicity of fluvial sediments downstream of the Ajka red mud spill,Hungary†

Orsolya Klebercz,*a William M. Mayes,b �Aron D�aniel Anton,a Vikt�oria Feigl,a Adam P. Jarvisc

and Katalin Gruiza

Received 21st February 2012, Accepted 31st May 2012

DOI: 10.1039/c2em30155e

An integrated assessment of biological activity and ecotoxicity of fluvial sediments in the Marcal river

catchment (3078 km2), western Hungary, is presented following the accidental spill of bauxite

processing residue (red mud) in Ajka. Red mud contaminated sediments are characterised by elevated

pH, elevated trace element concentrations (e.g. As, Co, Cr, V), high exchangeable Na, and induce an

adverse effect on test species across a range of trophic levels. While background contamination of the

river system is highlighted by adverse effects on some test species at sites unaffected by red mud, the

most pronounced toxic effects apparent in Vibrio fischeri bioluminescence inhibition, Lemna minor

bioassay and Sinapis alba root and shoot growth occur at red mud depositional hotspots in the lower

Torna Creek and upper Marcal. Heterocypris incongruens bioassays show no clear patterns, although

the most red mud-rich sites do exert an adverse effect. Red mud does however appear to induce an

increase in the density of aerobic and facultative anaerobic bacterial communities when compared with

unaffected sediments and reference sites. Given the volume of material released in the spill, it is

encouraging that the signal of the red mud on aquatic biota is visible at a relatively small number of

sites. Gypsum-affected samples appear to induce an adverse effect in some bioassays (Sinapis alba and

Heterocypris incongruens), which may be a feature of fine grain size, limited nutrient supply and greater

availability of trace contaminants in the channel reaches that are subject to intense gypsum dosing.

Implications for monitoring and management of the spill are discussed.

aDepartment of Applied Biotechnology and Food Science, BudapestUniversity of Technology and Economics, 1111 St. Gell�ert sq. 4,Budapest, Hungary. E-mail: klebercz.orsolya@gmail.com; Fax: +36-1-463-2347bSchool of Civil Engineering and Geosciences, Newcastle University,Newcastle upon Tyne, NE1 7RU, UK. E-mail: adam.jarvis@ncl.ac.uk;Fax: +44 (0)191 246 4961; Tel: +44 (0)191 246 4871cCentre for Environmental and Marine Sciences, University of Hull,Scarborough, YO11 3AZ, UK. E-mail: w.mayes@hull.ac.uk; Fax: +44(0)1723 370815; Tel: +44 (0)1723 357292

† Electronic supplementary information (ESI) available. See DOI:10.1039/c2em30155e

Environmental impact

The Marcal catchment (Hungary) was subject to the largest recorde

environment, in October 2010. We undertook sediment surveys in th

and biological methods. These highlight the adverse effect on num

despite the magnitude of the spill, the signal of the red mud is onl

underlying contamination of the system. This study provides valuab

accident of international importance and an industrial by-product

mental behaviour and toxicity.

This journal is ª The Royal Society of Chemistry 2012

Introduction

Numerous methods have been developed over the years to assess

the adverse impacts of pollution events in freshwater systems.

These range from direct physico-chemical testing and compar-

ison with consensus-based guidelines1,2 through to the use of

ecological indicators and bioassays at a wide range of trophic

levels from the benthic biota (e.g. diatoms and other bacteria,

algae and protozoa, invertebrates and macrophytes3) to assess

organism response. Comparison of the results of chemical

analyses and biological/ecotoxicological testing may also provide

additional insight, such as the gap between maximum potential

d release of caustic, metal-rich bauxite processing residue to the

e aftermath of the spill and applied a suite of physico-chemical

erous bioassays across the catchment. The data suggest that

y easily discernible at a small number of locations and suggest

le information on the response of instream biota to an industrial

for which there is minimal published information on environ-

J. Environ. Monit., 2012, 14, 2063–2071 | 2063

Fig. 1 Location map of sample stations.

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effect and the apparent observed adverse effects, which is basic

information typically included in risk-based management of

contaminated sites.3 In the European Union, the adoption of the

Water Framework Directive2 has set out measures for achieving

good chemical and ecological status in water bodies, and with

that provided integrated targets for instream biota, water quality

and sediment quality. Fluvial sediment quality guidelines,

however, are not as well developed as those for waters or

contaminated land. While a range of consensus-based guidelines

using the Threshold Effects Level (TEL) and Predicted Effects

Level (PEL) have been developed1,4 and are currently being

translated into formal guidelines across the EU, such thresholds

may be deemed too pessimistic in some settings when based on

partial or complete acid digestion.5 The adoption of extraction

procedures6,7 to improve bioavailability assessments and parti-

tion contaminants in different environmental phases aids such

assessments, but such approaches are limited by cost for exten-

sive surveys. As such, an integrated approach to monitoring and

assessing pollutants risks incorporating both chemical and bio-

logical tools has been espoused by numerous researchers8,9 and

regulators in recent years.

The release of 600 000–700 000 m3 of caustic (pH > 13) fine

fraction bauxite processing residue (red mud) suspension from

the Ajkai Timf€oldgy�ar Zrt alumina plant on the 4th October

2010 engulfed the villages of Kolont�ar, Devecser and

Soml�ov�as�arhely in western Hungary, causing widespread injury

and killing 10 people. Up to 40 km2 of urban and agricultural

land were inundated and downstream river systems were affected

by widespread fish kills as the caustic slug of material propagated

downstream.10–12 While there have been abundant studies on the

management and rehabilitation of bauxite residue deposits (see

recent reviews13–16), there have been relatively few studies on red

mud leachate chemistry17,18 and little on the geochemical

behaviour of red mud released in significant quantities in fresh-

waters. Furthermore, red mud contains many potential trace

contaminants (e.g. molybdenum and vanadium) that are not

particularly common in natural or contaminated land settings

and for which consensus-based environmental quality thresholds

are not well defined.1 In addition, few studies address the

potential mobilizing effect of the sodic leachant on contaminants

already present, which may be the main risk in some settings. As

such, the Ajka accident demanded an integrated approach to

monitoring the immediate and long term environmental impacts

of the spill on various receptors. Initial scientific studies from the

site have suggested that sodicity, as opposed to trace element

enrichment, is the key constraint to plant growth on red mud

affected floodplain sediments,12 while the hazard to human

health from fugitive red mud dusts has been suggested to be

equivalent to, or less than, that of urban dusts.19 Studies on the

fluvial sediment contamination have highlighted the abundance

of aluminium, vanadium, chromium, nickel, arsenic and rare

earth elements20 downstream of the spill, although the bulk of

these potential contaminants (with the exception of V) appears to

be associated principally with hard-to-leach residual phases that

are unlikely to be mobile in the environment.20,21 Speciation

studies have reinforced these patterns, but also highlighted the

prevalence of vanadium in mobile pentavalent species (the most

toxic form of V), which may be leached from red mud after

deposition on floodplain areas.22 Assessments of the fluvial

2064 | J. Environ. Monit., 2012, 14, 2063–2071

sediments have also highlighted the widespread signal of gypsum

dosing of stream waters, a control measure widely applied in the

immediate aftermath of the spill to neutralise the high alka-

linity.20,23 Although gypsum had a clear effect in lowering pH,

altogether more than 10 000 tonnes of gypsum settled alongside

red mud on the surface of fluvial sediment. As such, in places the

sediment was gypsum-dominated. These studies have thus

provided a detailed prognosis on the likely environmental risks

posed by the spill through invoking a range of physico-chemical

analytical techniques.

This study uses an integrated monitoring approach to assess

the biological activity and ecotoxicity of red mud-affected fluvial

sediments downstream of the Ajka spill in comparison with the

chemical–analytical results. Microbiological activity was char-

acterised by the number of colony forming cells, while toxicity

was measured at three trophic levels: microbial, plant (aquatic

and terrestrial) and animal toxicity using rapid, direct contact

laboratory bioassays. The integrated evaluation of the microbi-

ological, ecotoxicological and physico-chemical properties of the

sediments helps to identify the potential biological impacts of the

spill and infer drivers for any such impacts.

Materials and methods

Study site

Sample locations across the Torna Creek, Marcal River, R�aba

River and Mosoni Duna are shown in Fig. 1. Additional site

details and a description of the catchment can be found in Mayes

et al.20,21 The sites assessed by the integrated methodology here

comprise second order channels or higher from around the spill

site near Ajka to 110 km downstream near the confluence of the

Marcal–R�aba system with the Danube. A higher density of

sampling along the Torna Creek (T2–T6) was undertaken and

evenly spaced sample stations were situated along the course of

the Marcal River. Reference sediment samples were collected

This journal is ª The Royal Society of Chemistry 2012

Fig. 2 Integrated monitoring method used in the study.‡

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from upstream of the spill site in Ajka (T2), upstream on the

Marcal near Karak�o (M1) and prior to the confluence with the

Marcal on the R�aba (R1). These reference sites encompass

a range of sites covering the different land uses across the

3056 km2 catchment, from an industrialised urban headwater

(Ajka), a rural, agriculture-dominated headwater (M1) and

a mixed rural–urban catchment (R1).

Characterisation of sediment samples

The integrated monitoring methodology used in this study

consists of the determination of the physico-chemical properties

of the sediments and the measurement of the biological activity

and ecotoxicity of the sediment samples (Fig. 2).

Sampling and physico-chemical analyses

Four bulk (�500 g) sediment samples were collected at each

sample station by aggregating three randomly collected sub-

samples from a 12 m2 area of stream bed at each sample station.

Three of these were used for replicate chemical analysis, while

one was used for biological and ecotoxicological analysis. Sedi-

ments for chemical analysis were homogenized, air-dried, dis-

aggregated and sieved (2 mm aperture) in the laboratory prior to

microwave-assisted total digestion (aqua regia and HF)

following USEPA.24 Elemental concentrations in digests were

analysed using a Perkin Elmer Elan DRCII inductively Coupled

Plasma-Mass Spectrometer (ICP-MS; As, Cr and Mo) and an

Optima 5300 DV ICP-OES (all other elements). Exchangeable

elemental concentrations are taken from laboratory sequential

extraction procedures detailed in Mayes et al. (2011) and repre-

sent magnesium chloride (1 M at pH 7.0) extractable fractions.

Substrate pH was measured through the suspension of 10 g of

sample in 25 ml of distilled water. After shaking for 30 minutes at

200 rpm, pH was measured by pH electrode WTW Sentix 81

according to Hungarian Standard HS 21470/2-81. The dry mass

of 2 g wet sediment was measured after 24 h drying at 105 �Cuntil constant weight was achieved, and cooling in a desiccator.

‡ Details for individual sections reported elsewhere are referred to bysuperscript numbers: 1Mayes et al. (2011a), 2Mayes et al. (2011b), and3Burke et al. (2011).

This journal is ª The Royal Society of Chemistry 2012

Biological characterisation and ecotoxicity testing

Aerobic heterotrophic colony forming units. For the measure-

ment of microbial activity (living cell concentration) 1 g wet

sediment was placed into 10 ml tap water (sterilized in an auto-

clave, 10 min at 121 �C) and was shaken for 30 minutes at

400 rpm (3 replicates). A 10 fold dilution series was prepared and

100 ml of the 101, 102 and 103 dilutions were measured into Petri-

dishes. 10 ml of meat agar (cooled to 45 �C, composition: 3 g

meat extract, 5 g glucose, 5 g peptone, 0.5 g NaCl, 17 g agar, 1 l

distilled water, sterilized for 10 min at 121 �C) was poured into

each Petri-dish and was incubated at 30 �C for 48 hours.25 The

number of colonies was counted.

Vibrio fischeri luminescence inhibition test. Two grams of dry

sediment was suspended in 2 ml 2%NaCl solution and a five-step

dilution series was prepared. After the measurement of the

reference luminescence intensity, 50 ml of the dilution series was

added to the test medium. The luminescence intensity was

repeatedly measured after 30 min exposure time with a lumin-

ometer (Lumac Biocounter M 1500 1). The toxicity was char-

acterised by the inhibition rate (%) of the samples and the

copper-equivalent EsD50 CuEq (mg/kg) (Cu equivalent concen-

tration of an unknown contaminant or mixture of contaminants

in the sediment sample causing 50% inhibition). Sample T2 taken

from upstream of the spill area was used as a reference sediment,

when inhibition rate was calculated in %, because this sediment

was not toxic to Vibrio fischeri at all.26

Sinapis alba root and shoot growth inhibition test. For the

Sinapis alba (white mustard) test, the method of Leitgib et al.26

was adopted, where 5 g of air dried sediment was measured into

a Petri-dish, wetted with 3.5 ml water and 20 seeds were placed

on top. The samples were incubated at 25 �C for three days. The

lengths of roots and shoots were measured manually. Root and

shoot growth inhibition was calculated: I(%) ¼ (C� P)/C� 100,

where I: inhibition%; C: length of roots/shoots on uncontami-

nated control (tap water on a filter paper); P: length of roots/

shoots on polluted sample.

Lemna minor leaf reproduction inhibition test. 10 g dry mass-

equivalent quantities of wet sediment samples were put into

standard 150 ml glasses (d ¼ 5.8 cm), and 20 ml Lemna minor

(Hoagland) nutrient solution was measured on the top. After the

suspension had cleared, 10 seedlings of Lemna minor were placed

into the solution. The samples were incubated at 21 �C during 8

hours dark and 16 hours illuminated cycles. After the 7th day the

duckweed leaves were air dried, and after 24 hours of extraction

in 5 ml 96% ethanol, chlorophyll content was measured spec-

trophotometrically (Sanyo SP55 UV/VIS, at 470, 649, 664 nm).

Chlorophyll content was calculated as follows: C ¼ (5.24A664 +

22.24A649) � 5/mduckweed. Inhibition% was calculated compared

to negative control (plants grown without sediment, only on

nutrient solution), and to headwater sediment samples

(T2, M1).27

Heterocypris incongruens mortality test. Heterocypris incon-

gruens is a sediment-dwelling ostracod living in sediments widely

used in toxicity testing.28 Using Ostracodtoxkit F�, 1.5 g dry

J. Environ. Monit., 2012, 14, 2063–2071 | 2065

Fig. 3 Principal Component Analysis of sediment elemental composi-

tion data (after Mayes et al., 2011).x

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mass-equivalent quantities of wet sediment samples were put into

10 ml glasses (d ¼ 3.7 cm), and 8 ml standard mineral water

solution was measured on the top. After the suspension had

cleared, 10 specimens of Heterocypris incongruens were placed

into the solution. The living specimens were counted for 3 days.

10 ml of standard solution without sediment was used as negative

control. Inhibition% was calculated from the number of living

animals in sediment samples compared to the negative control.

Statistical methods. Data were not normally distributed even

after log transformation (Kolmogorov–Smirnov: p < 0.05),

therefore non-parametric methods are used. Data were stan-

dardized prior to Principal Component Analysis. All analyses

were undertaken in Minitab v. 15.

Results and discussion

Physico-chemical patterns

The composition of red mud from the spill at Ajka has been

shown to be relatively consistent with constituents from previous

analyses.12,19,21 The samples assessed here were taken from across

the impacted catchments and presented a mixing gradient of

unaffected fluvial sediments with red mud and gypsum. These

gradients are shown graphically in Fig. 3, and highlight the

x Ordination of sample sites by the first two principal components in theupper image. Variable loading on the first two principal components inthe lower image. ‘Ex_TM’ ¼ exchangeable trace elements (¼ SAs CuCo Cr Cd Mo Ni V Zn); ‘Ex_Al’ ¼ exchangeable aluminium;‘Ex_Na’ ¼ exchangeable sodium. M2a, M2b, and M2c are replicatesfrom the same sample station.

2066 | J. Environ. Monit., 2012, 14, 2063–2071

samples enriched with red mud to the right hand side of the plot.

These are characterised by elevated total Na, Al, Fe and various

trace (e.g. Co, Cr, Cu, Ni, V) and rare earth elements (Fig. 3 (ref.

21 and 22)). The most red mud-enriched samples are seen to be

downstream sites T6 and M2 (Table S1†) where the high velocity

channelised reaches of the upper Torna Creek give way to lower

gradient, lower velocity reaches more conducive to sediment

deposition.21

Here, concentrations of Cr, Ni and As exceed both TEL and

PEL thresholds, while concentrations of V and Co exceed

‘background’ levels.1 Concentrations of Cr, Ni and V at M2 and

T6 lie between 34 and 62% of those reported for source material

(sample K1, which represents red mud at the spill site21), which

offers an insight into the relative mixing of the red mud source

term with trace element-poor unaffected sediments. PC1 repre-

sents this gradient of dilution of red mud dominated samples (e.g.

T6) towards reference sites in the left hand side of the plot (e.g.

M1, T2). PC2 appears to be dominated by the signal of gypsum

dosing, where enrichment with Ca and S characterises those

samples particularly from the lower Torna Creek and Marcal

River (M7, M11) where extensive smothering of benthic zones

with gypsum and secondary carbonate deposits23 was apparent.

Interestingly, exchangeable aluminium and exchangeable total

trace element concentrations correlate well with areas of exten-

sive gypsum dosing. This may be a feature of uptake of mobile

chemical species from the water column by extensive secondary,

amorphous carbonate deposits,23 which can be an efficacious

sink for various trace elements.29–31

Reference samples (T2, M1 and R1) plot closely with down-

stream, largely unaffected, sites (e.g. MD1, R2) and are charac-

terised by enrichment of Ba and K, indicative of Triassic

dolomite-dominated bedrock in the catchment.11,21 Some of the

reference samples also show modest elevations in trace elements

above quality thresholds.21 Enrichment of Co, Cr, Ni and V at

MD1 is more likely to reflect long term exposure of these

downstream reaches to other industrial sources around the town

of Gy}or than red mud itself given the low Na and Fe concen-

trations at these sites. Substrate pH varied between 8.14 and 9.88

across the sites. Exchangeable Na correlates positively with

substrate pH (rs ¼ 0.61, p ¼ 0.007), suggesting that residual

NaOH in the red mud-affected sediments elevates pH above the

range considered optimal for aquatic life (pH 6–9).

Aerobic heterotrophic colony forming cell-concentration

The counts of colonies of aerobic heterotrophic bacteria offer an

indication of the microbial activity of the fluvial sediments.

Reference samples report a range in microbial activities with

colony counts between 34.8 million cells (T2) and 848.4 million

cells (R1). The lower value at T2 may reflect other industrial

pollution sources around the town of Ajka, while the higher

organic content of larger rivers (R�aba at R1) may contribute to

higher microbial activity. Long term monitoring data from the

rivers suggest that this is the case with mean biological oxygen

demand (BOD) in the R�aba (2.8 mg l�1) and Mosoni-Duna

(2.9 mg l�1) being twice that of the Marcal (1.4 mg l�1).32 The

effect of red mud appears to stimulate aerobic heterotrophic

microbial populations, with a 10–100 times increase in colonies

over reference samples. This pattern is reinforced by statistically

This journal is ª The Royal Society of Chemistry 2012

Fig. 4 Relationship between bacterial activity and substrate pH (left) and exchangeable Na (right) across the Marcal and R�aba catchments.

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significant positive correlations between colony counts and

substrate exchangeable Na (rs ¼ 0.66, p ¼ 0.003), pH (rs ¼ 0.51,

p¼ 0.03) and elements indicative of red mud enrichment, such as

Cr, Fe and Ni (Fig. 4 and Table S1†). Studies on the amendment

of metal-contaminated soils with red mud have found similar

findings. Amendment of such soils with 2–10% volume red mud

has the effect of substantially increasing microbial biomass33 and

is thought explicable due to diminished availability of toxic

metals.34 Although Lombi et al.33 suggest lesser Cu availability

with red mud amendment as the key driver for increased

microbial activity, it is difficult to speculate in this case given the

range of trace elements that are elevated in the samples.21

Certainly, the equivalent mixing rates in this study far exceed

10% red mud by volume for the most contaminated sites (T6,

M2), without there being strong inhibitory effects. Results do

suggest however that the added gypsum may have greater

adverse effect on sediment microbiology than the red mud itself.

The gypsum-rich samples (as indicated by elevated S: e.g. M10,

M11) report lower cell numbers than adjacent sample sites with

lower S concentrations, although no statistically significant

correlation was found between S and colony numbers. The

smothering of the streambed with fine grained gypsum and

associated secondary carbonate deposits may play an important

physico-chemical role in limiting oxygen diffusion through the

substrate and enhancing anoxia in smothered sediments. If this

were the case, a sufficient shift in redox away from conditions

suitable for aerobic bacteria and facultative anaerobes to those

only suitable for anaerobes could explain the diminished aerobic

microbial activity at the gypsum affected sites. Physical

constraints to biological populations have been highlighted at

similar hyperalkaline sites affected by carbonate crust deposi-

tion.31,35 However, assessment of microbial response in streams

affected by such deposits has not been studied and warrants

further attention.

Vibrio fischeri luminescence inhibition test

The luminescence inhibition of Vibrio fischeri in sediments is

calculated from a serial dilution of sediment suspension. The

shadowing effect of the suspended sediment is corrected with the

use of uncontaminated control sediment, which in this case is

sample T2. 50% effective sediment dose (EsD50) is calculated by

fitting a dose–response curve on the different inhibition

percentages of the dilution series. This is expressed in Table S2†

showing:

This journal is ª The Royal Society of Chemistry 2012

a. EsD50 sediment mg s. In this case, the smaller the number,

the more toxic is the sample.

b. In Cu-equivalent, comparing the inhibition percentage

of the sample with that of a standard Cu concentration ser-

ies. In that case, the greater the number, the more toxic is the

sample.

With the exception of the T2 and M1 reference samples and

mid-course samples from the Marcal (M6 and M10) the sedi-

ments were very toxic to them: all the EsD50 values are low, and

mostly in the same range. These include the R1 reference sample

along with downstream sample sites on the R�aba (R2) and

Mosoni Duna (MD1), where the signal of red mud is difficult to

discern in the physico-chemical data21,22 (Fig. 3). This suggests

that other background sources of pollution in the catchment are

contributing to the response seen here. However, for the Cu

equivalent data (Table S2†), there are relatively weak, yet

significant positive correlations between sample toxicity and

various red mud indicators such as exchangeable Na (rs ¼ 0.53,

p ¼ 0.022), total Co, Cr, Ni and V (Table S3†). These do tend to

be skewed by the red mud-enriched M2 sample (Fig. 5), which

reported the most toxic effects on the testorganism. Vibrio

fischeri has been shown not to be the most sensitive testorganism

for elevated Al36 and salinity37 but does show sensitivity to

various trace elements36 with toxicity apparent in studies on

similar wastes elsewhere containing a complex of trace elements

such as coal combustion residues.38 However, while there are

significant correlations with various red mud-sourced contami-

nants it is impossible to discern individual variables, or groups of

variables, that are exerting the adverse effect on V. fischeri given

that it is such a sensitive testorganism. Another factor that

requires consideration is grain size, with fine particle size known

to be an important confounding factor in the Vibrio fischeri

test.39,40 The fine particle size of the red mud at the site has been

previously documented20 with the most impacted sites (M2, T6)

representing a mixture of the silty-sand sediments that charac-

terise reference sites mixed with red mud which lowers median

particle size. Similar physical factors may be responsible for the

observed response at the gypsum contaminated sites where grain

size was finer than reference conditions.

Lemna minor leaf reproduction inhibition test

The Lemna minor test supports the results of the other ecotox-

icological tests: it can be seen clearly that red mud in Torna Creek

samples (T3–6) caused 10–30% inhibition, while in the most

J. Environ. Monit., 2012, 14, 2063–2071 | 2067

Fig. 5 Response of Vibrio fischeri (Cu-equivalent EsD50) with total Al, Co, V and exchangeable Na. Linear regression lines are only provided where

correlations are significant at p < 0.05.

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contaminated Marcal sample station (M2), more than 60% inhi-

bition occurred. In the other measurement points of Marcal,

where red mud is not present in appreciable quantities, toxicity

remains under 20%, with gypsum dosing having no noticeable

effect compared to reference samples. The high toxicity in the

R�aba (R2) and Mosoni-Duna (MD1) samples is most likely

Fig. 6 Lemna minor inhibition percentage relative to experim

2068 | J. Environ. Monit., 2012, 14, 2063–2071

a diffuse pollution signal from decades of industrial activity and

municipal sources in the area of Gy}or city,41 which may include

organic contaminants not measured here. There are highly

significant positive correlations between Lemna minor inhibition

rate and exchangeableNa (rs¼ 0.69, p¼ 0.002), Co,Cr, Fe andNi

(rs between 0.70 and0.75; p<0.001;Fig. 6) and significant positive

ent control against key red mud-controlled parameters.

This journal is ª The Royal Society of Chemistry 2012

Fig. 7 Sinapis alba root and shoot inhibition against exchangeable trace element and aluminium content of the sediments.

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correlations with pH (rs ¼ 0.49, p ¼ 0.04) and V

(rs ¼ 0.49, p ¼ 0.03). This suggests that red mud presence is

amajor control onL.minor growth in this study and thatL.minor

is a useful biomonitor for the impact of red mud contamination

across the downstream catchments. It is however impossible to

suggest causal links between individual parameters and L. minor

response. L. minor is known to be sensitive to pH, with increased

mortality above pH 8 documented by some researchers.42 Lem-

naceae are also documented to be sensitive to elevated metal

concentrations present in this study such as Ni43 and V.44

Heterocypris incongruens mortality test

The inhibition of growth of Heterocypris incongruens in the refer-

ence samples is quite variable and relatively high (range 10–60%).

The most toxic sample with regard to H. incongruens mortality is

M2 (Table S2†), however, there were no significant correlations

between H. incongruens mortality and key red mud sediment

indicators (e.g. trace elements,Al andNa: Table S1†; Fig. 3). There

is also slightly elevated mortality at other red mud affected sites

(e.g. T3, T4) and gypsum-affected sediments (M7–M11) above

reference sites. Most of the downstream samples between heavily

red mud-affected and gypsum affected sediments show relatively

low toxicity (M4–M6). H. incongruens is known to be a sensitive

testorganism for metal ions in direct contact tests45 and is also

known to be reasonably tolerant of brackish conditions.46As such,

it may be the elevated trace element concentrations as opposed to

elevated salinity at the most impacted sites that are controlling

response. However, if there are toxic effects induced by red mud,

there are no clear general trends and H. incongruens response

cannot be relatedwith confidence to particular contaminants from

the data collected here. Similar to other tests, the fact that some

reference sites report adverse effects (T2, R1) suggests that other

pollutant sources (beyond red mud contamination), not measured

in this study, are likely to be present, particularly in the urban/

industrial reaches of the catchment.

Sinapis alba root and shoot inhibition test

Seed germination and early growth tests utilising Sinapis alba

have been widely used in ecotoxicological testing,47,48 providing

This journal is ª The Royal Society of Chemistry 2012

a useful test species in this case for terrestrial habitats (e.g.

floodplains) affected by the red mud. Table S2† shows the root

and shoot inhibition percentage in test specimens relative to

laboratory control specimens. It is clear that even within refer-

ence samples (T2, M1, R1), there is a reasonable degree of

variability, albeit inhibition of shoot and root growth is relatively

low. Consistent with the other tests, the most red mud-affected

sites (T6 and M2) and heavily gypsum affected sites (M7–M11)

report the most adverse response. There are significant positive

correlations between root inhibition percentage and exchange-

able trace elements (which are high in both red mud-rich and

gypsum dosed sites: Fig. 3), exchangeable Al and total As, Cd

and V (Table S3†). Significant positive correlations are also

apparent between shoot inhibition percentage and exchangeable

trace elements, exchangeable Al and total Al (Fig. 7). These

patterns are consistent with the reported low tolerance of S. alba

to Al49 and its bioaccumulation of trace elements which may

impede growth.47 However, other factors such as soil physical

structure and redox condition (given it is a terrestrial species)

may be also considered, particularly at sites inundated with

a thick red mud layer where anoxic conditions may contribute to

the patterns seen here.

Comparative evaluation of the sensitivity of the applied

testorganisms

For comprehensive evaluation of the sensitivity of the tes-

torganisms applied for the toxicity testing of sediments

contaminated with red mud and gypsum, the samples were

divided into clusters according to concentrations of specific

indicator elements. Samples with exchangeable Na over

600 mg kg�1 were classified as red mud contaminated, and S over

2000 mg kg�1 as gypsum contaminated sediments. The remaining

samples were classified as references (unaffected by the disaster,

originated from upstream sampling points for each river), and

moderately contaminated samples (comprising mixed sediments

with concentrations of S and exchangeable Na below the

thresholds stated above).

All toxicity data were indexed according to their relative

toxicity compared to the most toxic sample, by each

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testorganism. The average relative toxicity index of each cluster

of samples was pictured in a radar chart (Fig. 8).

In total, all testorganisms showed higher toxicity for red mud

contaminated samples than for all the others, while reference

samples showed the lowest toxicity in all four tests. Toxicity of

gypsum contaminated samples to Sinapis alba and Heterocypris

incongruens was similar to the red-mud contaminated ones. Both

of these testorganisms are in close interaction with the sediment

during testing, while Vibrio fischeri and Lemna minor – showing

lower toxicity – live in the water phase, exposed only to dissolved

aqueous contaminants. The fact that the samples contaminated

by fine grained gypsum caused greater inhibition on testorgan-

isms, which are continuously in direct contact with the sediment

during a longer time period (3–5 days), supports the notion that

besides the chemical composition, the physical nature of the

substrate also influences the toxicity.

Conclusions and management implications

The suite of biological and ecotoxicity tests undertaken in this

study has highlighted a broad range in response across the

catchments impacted by the red mud spill at Ajka. The sample

stations most affected by the spill exhibit an adverse effect across

a range of trophic levels, although the red mud appears to have

a stimulatory effect with regard to microbial activity. While

composition in aerobic microbial communities was not assessed

here due to logistical reasons, there may have been changes in the

community structure itself at some sites in response to the spill.

Lemna minor appears to be a particularly sensitive testorganism

to the effects of red mud deposition and could provide a useful

biomonitor to assess future system recovery. The physico-

chemical patterns in stream sediment contamination (see Mayes

et al.20,21) highlighted hotspots of deposition downstream of the

site (e.g. T6, M2), which is a pattern mirrored in the biotic

response. Given the cocktail of trace elements present in the red

mud, and concomitant elevations of exchangeable Na and Al, it

is difficult to infer specific controls on the biological response

across the sample sites. Furthermore, residual pollution releases

from other contemporary and historic industrial activity in the

catchments also appear to add noise to the observed responses.

The relatively limited spatial extent of such red mud-enriched

hotspots and associated biological impacts is, however, encour-

aging for system recovery. Given this, expansion of red mud

Fig. 8 Comparative evaluation of the sensitivity of the testorganisms.

2070 | J. Environ. Monit., 2012, 14, 2063–2071

removal operations, which have been extensive for floodplain

deposits in the aftermath of the spill, to instream dredging is

unlikely to be of major benefit in terms of volumes of material

removed. Furthermore, such operations may lead to the risk of

remobilisation of other potential contaminants previously

sequestered in fluvial sediments (e.g. mercury and organics) that

have not been measured in this study, but are likely to be present

given the industrial history of the catchment and the adverse

responses apparent for some bioassays at reference sites. Further

evaluation of the long term exposure of sediment dwelling

organisms in the system to other pollutant sources (i.e. beyond

the red mud) would be beneficial in providing a more detailed

evaluation of such management options.

The extensive pollution of stream sediments with gypsum in

the Marcal River also appears to exert a negative biotic response

with regard to Sinapis alba and Heterocypris incongruens in

particular. Whether the adverse effect of gypsum-rich substrates

represents a feature of physical smothering, nutrient deficiency,

poor physical structure of the substrate or availability of trace

contaminants is unclear. Such issues have been highlighted at

other sites impacted by extensive calcareous deposits.31,35,50,51

Given that gypsum dosing was undertaken over a relatively

limited time period, reasonably rapid recovery of the system

would be anticipated subsequently compared to systems exposed

to ongoing deposition.

Acknowledgements

Biological and ecotoxicological experiments were carried out as

part of SOILUTIL project, funded by the Hungarian National

Technology Program’s Liveable and Sustainable Environment

sub program under the code number TECH 09 A4. We are

grateful to the UK Natural Environment Research Council for

funding the physico-chemical analysis (grant NE/I019468/1) and

to Bob Knight (University of Hull) and Jane Davis (Newcastle

University) for undertaking them. We also thank Gy}oz}o Jord�an

(Geological Institute of Hungary), Kovacs Laszlo and Istv�an

Csonki (Central Danubian Water and Environment Authority)

for site information and access.

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