ECOTOXICOLOGY ANDENVIRONMENTALSAFETY 17,105-l ll(l989)

The Effect of Humic Acid on the Toxicity and Bioavailability of Trivalent Chromium

R.ALLANSTACKHOUSEANDWILLIAM H. BENSON'

Toxicology Program, College ofPharmacy and Health Sciences, Northeast Louisiana University, Monroe, Louisiana 71209-0470

Received April 18, 1988

The influence of humic acid (HA) on the toxicity and bioavailabiity of two forms of trivalent chromium (chromic chloride and chrome lignosulfonate) was evaluated using a common fresh- water invertebrate, Daphnia pulex. With both compounds, the 50 mg/liter HA significantly decreased toxicity at all time points examined. The remaining two HA concentrations, 0.5 and 5 mg/liter, either had no influence or decreased the toxicity of the compounds. Humic acid appeared to have no influence on the bioavailabihty of chrome lignosulfonate. However, for chromic chloride, 5 and 50 mg/liter HA decreased the percentage free chromium at all time points examined. 0 1989 Academic Press, Inc.

INTRODUCTION

Chromium is one of a few trace elements essential for life and serves as an impor- tant raw material for technology, while also presenting problems of toxicity to man and other life forms (Mertz, 1982). Many industries including metallurgical, refrac- tory, chemical, tanning, and oil exploration use chromium as a raw material (Langard and Norseth, 1986). Chromium-containing wastes are often disposed of by these in- dustries to the land surface or as a part of a complex aqueous solute mixture to waste ponds or lagoons (Zachara et al., 1987). These practices can result in the migration of, and contamination by, chromium to nearby groundwater. The preliminary find- ings of the priority pollutant monitoring project of the Nationwide Urban Runoff Program (NURP) indicated that chromium was a frequently detected (57% of sam- ples) pollutant with aqueous levels ranging from 1 to 34 pg/liter (Cole et al., 1984). Perhaps the most catastrophic incidence of chromium toxicity resulted from the de- position of approximately 530,000 tons of untreated slimes and factory wastes by the Nippon Chemical Industrial Co. around Tokyo, Japan. Levels of chromium reached 2000 times the official threshold limit in groundwater near the waste sites and resulted in 30 human mortalities (Wittmann, 1983).

Chromium exists in two primary oxidation states, hexavalent, Cr(VI), and triva- lent, Cr(III), although it can exist in valence states from 2- to 6-t (Meams et al., 1976). In nature, chromium is found almost exclusively as Cr(III), whereas Cr(V1) primarily results from anthropomorphic activities (Mertz, 1982). Hexavalent chro- mium is stable in most natural waters and its toxicity is not affected by hardness, while Cr(II1) readily complexes with organic and inorganic compounds and its toxicity is significantly influenced by hardness (U.S. EPA, 1980). In most organisms, Cr(V1) is

’ To whom correspondence should be addressed at Department of Pharmacology, School of Pharmacy, University of Mississippi, University, MS 38677.

105 0147-65 13/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

106 STACKHOUSE AND BENSON

approximately 100 times more toxic than Cr(II1) (Wittmann, 1983). Although nu- merous studies have been conducted with both Cr(V1) and Cr(II1) to determine the acute toxicity to freshwater organisms, there is a paucity of relevant data on the influ- ence of dissolved organic compounds (DOCs) on the biological effects of chromium.

Total metal concentration can be a misleading indication of the bioavailability of a metal due to chemical speciation (Leland and Kuwabara, 1985). Several investigators have reported that the ionized or “free” form of the metal is responsible for the ob- served toxicity (Allen et al., 1980; Hongve et al., 1980; Giesy et al., 1983). Dissolved organic compounds (e.g., humic (HA) and fulvic acids) as well as inorganic materials in natural waters can affect speciation of metals and hence toxicity (O’Donnel et al., 1985). The sorption of metals to organic and inorganic compounds occurs in all aquatic systems and is perhaps the single most important environmental process affecting fate (Baughman and Lassiter, 1978). Therefore, the objective of the present investigation was to determine the influence of humic acid on the toxicity and bio- availability of Cr(II1).

MATERIALS AND METHODS

Culture and Test Conditions

Laboratory cultures of Daphniapulex were obtained from the U.S. Environmental Protection Agency, Environmental Research Laboratory, in Duluth, Minnesota. In our laboratory, the daphnids were cultured in moderately hard reconstituted water and fed green algae, Selenastrum capricornutum, and a cerophyll, yeast, and trout food mixture daily (U.S. EPA, 1985a).

All tests were conducted in aged, moderately hard reconstituted water previously filtered through activated charcoal to remove endogenous DOCs, as well as through a 0.45~pm membrane filter to remove the remaining particulates. Dilution water characteristics were as follows: alkalinity, 73 + 12 mg/liter CaC03; hardness (EDTA), 92 + 8 mg/liter CaC03; and pH, 8.0 + 0.1. The HA (Aldrich Chemical Co.) was prepared by dissolution and centrifugation at 6000g for 30 min, as well as filtration through a 1.2~pm membrane hlter to remove particulates. Trivalent chromium was delivered as CrC13.6H20 and chrome lignosulfonate. Chrome lignosulfonate is a form of Cr(II1) used as a deflocculent for thinning water-based drilling muds, in addi- tion to maintaining the mud in a fluid state (U.S. EPA, 1985b). The pH of the stock solutions of both CrQ and chrome lignosulfonate was adjusted with NaOH to 8.0.

Acute Bioassays/Bioavailability

The influence of HA on the toxicity and bioavailability of the chromium species was assessed using modifications of methods previously described (Stackhouse and Benson, 1988). Briefly, the acute toxicity of the metals was determined by static me- dian lethal concentration (L&J bioassays at 48,72, and 96 hr in the presence of four HA concentrations (0, 0.5, 5, and 50 mg/liter). However, due to precipitation, the acute toxicity of CrC& was determined using static renewal LCso bioassays with metal solutions renewed every 24 hr. The bioavailability of the chromium species was eval- uated by determining the decrease in percentage unbound metal (96-hr LCso value of the HA control) with time (3,6, 12,24,48, and 96 hr) in the presence of the four HA concentrations.

HUMIC ACID AND TRIVALENT CHROMIUM 107

mg,‘l HA

mg/l HA

mg/l HA

d z

&8 50.0 mg/l HA

10000

z

B 1000

2

G

j$ 100

2

48 72 96 TIME (hr)

FIG. 1. Influence of humic acid on the median lethal concentration of chromic chloride to Duphnia pulex. *Significantly different from simultaneous control (0.0 mg/liter HA) at P < 0.05.

Chemical Analysis

Aqueous metal concentrations were determined using a Perkin-Elmer Model 3030B atomic absorption spectrophotometer equipped with a Model HGA-600 graphite furnace by the modified methods of Arpadjan and Krivan ( 1986). The exper- imental concentrations of HA were determined by the UV spectrophotometric method of Zitko et al. (1973) using a Gilford Response UV-Vis spectrophotometer.

Data Analysis

Median lethal concentrations (L&,‘s) and 95% confidence limits were calculated by probit analysis (SAS Institute Inc., 1985) or by the moving angle method (U.S.

200 LEGEND

48 72 TIME (hr)

96

FIG. 2. Influence of humic acid on the median lethal concentration of chrome lignosulfonate to Daphnia pulex. *Significantly different from simultaneous control (0.0 mg/liter HA) at P -c 0.05.

108 STACKHOUSE AND BENSON

1201 1 LEGENO

1 m 0.0 mg/l HA

100 m 0.5 mg/l HA

g

5 0

5O:O

mg/l HA

5 60 mg/l HA

5 &

8 60

e E M 40

20

n 0 3 6 12 24 40 96

TIME (hr)

FIG. 3. Influence of humic acid on the percentage free chromium with time as determined by dialysis studies. Metal concentration of the spike of chromic chloride was equal to 98.2 pg Cr/liter.

EPA, 1985a). Significant differences between L&‘s, obtained from the bioassays, were assessed by the standard error of the difference (Sprague and Fogels, 1977). Differences were considered significant at P < 0.05.

RESULTS

Acute Toxicity

With both &Cl3 (Fig. 1) and chrome lignosulfonate (Fig. 2), the 50 mg/liter HA significantly decreased toxicity at all time points examined. In addition, the toxicity of G-Cl3 was significantly decreased by the 5 mg/iiter HA concentration by 72 and 96 hr and the 0.5 mg/liter HA by 96 hr. For chrome lignosulfonate, the 5 mg/liter HA had no significant influence on toxicity, while the 0.5 mg/liter HA significantly decreased toxicity by 48 hr but not at the remaining time points.

Bioavailability

Humic acid had a greater influence on the bioavailability of CrCl, (Fig. 3) than chrome lignosulfonate (Fig. 4). The 5 and 50 mg/liter HA decreased free chromium to a maximal extent by 24 hr with the 5 mg/liter HA concentration decreasing free chromium to 27%, while the 50 mg/liter reduced it to 18%. At none of the time points examined did the 0.5 mg/liter HA decrease free metal. Humic acid had no influence on the bioavailability of chrome lignosulfonate. The observed lack of influence could be a result of the molecular weight cutoff ( 1000 Da) of the dialysis membrane used in this investigation. Since the lignosulfonate molecule is a polymeric lignin derivative, containing large numbers of sulfonic acid, carboxylic acid and phenolic groups to which the chromium is bound (Duke, 1984), the membrane would exclude the ligno- sulfonate molecule, as well as the HA. By comparing the LCsO value (90.4 mg Cr/liter by 96 hr for the HA control) for chrome lignosulfonate with the LCX, for CrC& (98.2 pg Cr/liter by 96 hr for the HA control), it is apparent that only a small fraction of the

HUMIC ACID AND TRIVALENT CHROMIUM 109

120

2c

C

, --

,- 0 3 6 12 24 48 96

TIME (hr)

LEGEND

0 0 mg/l HA

ii 0:5 mg,‘l HA

m 5.0 mg,‘l HA

&8 50.0 mg/l HA

FIG. 4. Influence of humic acid on the percentage free chromium with time as determined by dialysis studies. Metal concentration of the spike of chrome lignosulfonate was equal to 90.4 mg Cr/liter.

total chromium results in the observed toxicity, while the majority of the chromium is bound to the lignosulfonate molecule. It is likely that the dialysis method used was unable to detect any binding of the free chromium fraction by the HA.

DISCUSSION

When comparing with results previously obtained in our laboratory (Stackhouse and Benson, 1988), HA had a greater influence on the toxicity of the Cr(II1) com- pounds than on that of the Cr(V1). The 50 mg/liter HA concentration significantly decreased the toxicity of both Cr(II1) compounds at all time points examined, while only significantly decreasing toxicity by 48 hr for Cr(V1). In addition, the 5 mg/liter HA decreased the toxicity of 0-Q by 72 and 96 hr. Also, with the exception of chrome lignosulfonate, HA had a greater influence on the bioavailability of Cr(II1) than on that of Cr(V1). Previously, HA was observed to have little influence of the bioavailability of Cr(V1) with maximal decreases of 2 and 7.5% free Cr(V1) by 5 and 50 mg/liter HA, respectively (Stackhouse and Benson, 1988). However, for CrC&, percentage free chromium was maximally decreased by 73 and 82% with 5 and 50 mg/liter HA, respectively. These results are consistent with the findings of the U.S. EPA ( 1980) indicating that, in general, Cr(II1) is significantly influenced by inorganic and organic compounds, while Cr(V1) is not.

The most likely explanation for the influence of HA on the biological effects of Cr(II1) and Cr(V1) relates to the speciation of chromium in freshwater. For the dilu- tion water used in this investigation, the thermodynamic data would indicate that the dominant dissolved species for Cr(V1) would be CrO-, while Cr(II1) would exist as Cr(OH)2+ (Cranston and Murray, 1980). Humic acid generally binds to metals by removal of a proton from carboxylic acid or hydroxyl functional groups followed by coordinate bonding to the metal (Giesy and Alberts, 1982). This binding mechanism would indicate that HA could bind readily to a cation (e.g., Cr(III)), but not to an

110 STACKHOUSE AND BENSON

anion (e.g., Cr(V1)). Anionic compounds have been demonstrated to bind weakly to HA through H-bonding (Stevenson, 1972).

CONCLUSIONS

Understanding the behavior of trace metals in the environment is essential for ac- curate prediction of the toxicity of metals in freshwater ecosystems. The interaction of trace metals and DOCs, such as HA, can result in the alteration ofthe physiochemi- cal form ofthe metal, which in turn may affect bioavailability and toxicity. The results of this investigation indicate that the influence of HA on the biological effects of chromium is dependent on (i) oxidation state, (ii) temporal relationship, and (iii) HA concentration.

ACKNOWLEDGMENT

This research was supported by the School of Pharmacy, an operational unit of the College of Pharmacy and Health Sciences, Northeast Louisiana University.

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