Chromium trioxide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1959 to 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The EU RAR summarises the results of a number of studies which individually have limitations regarding study design and reliability. However, the results of these studies when taken as a whole, provide an adequate assessment of toxicity to micro-organisms. The data are considered to fulfil the criteria laid down in Annex XI to Regulation 1907/2006: adequate for classification and labelling, adequate coverage of key parameters (inhibition), exposure duration comparable or longer that Article 13(3) methods, adequate documentation provided: EU RAR, of which parts have been copied to this endpoint record. Reliability for endpoint also increased based on test results from several species.

Qualifier:

no guideline followed

Guideline:

other: Range of tests reported

Principles of method if other than guideline:

Several studies reported with different methods.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Range of tests reported

Vehicle:

no

Details on test solutions:

Range of tests carried out

Test organisms (species):

other: Range of microbes reported

Details on inoculum:

Data from several studies

Test type:

other:

Water media type:

freshwater

Limit test:

no

Remarks on exposure duration:

Data from different tests

Post exposure observation period:

Not reported

Hardness:

Data from different tests reported

Test temperature:

Data from different tests reported

pH:

Data from different tests reported

Dissolved oxygen:

Data from different tests reported

Salinity:

Not applicable

Nominal and measured concentrations:

Data from different tests reported

Details on test conditions:

Data from different tests reported

Reference substance (positive control):

no

Details on results:

Ross et al. (1981) reported that an influent concentration of 10 mg Cr (VI)/l reduced the efficiency of a model activated sludge plant by 5%, as measured by effluent chemical oxygen demand. The same or similar results were also found by Barth et al. (1967) using a pilot scale activated sludge sewage treatment plant. In this study, chromium (VI) (form unknown) was added into the influent to the sewage treatment plant, and the plant was allowed to acclimate for 2 weeks before data on the functioning of the plant were obtained. The plant was then run for a further 60 days and the quality of the final effluent from the plant (in terms of biological oxygen demand (BOD), chemical oxygen demand (COD), turbidity and suspended solids) was compared
with that from an identical plant receiving domestic sewage only. It was found that once above a threshold concentration where the efficiency of the treatment plant was affected, there was no further deterioration in performance of the plant until very high concentrations of chromium (VI)
were reached. The threshold concentration of chromium (VI) identified in the study was 10 mg Cr/l as a continuous concentration in plant influent. The study also investigated the effects of pulses of higher concentrations of chromium (VI) on the treatment plant. It was found that a
chromium (VI) concentration of 500 mg/l (as a four hour dose) in influent had no noticeable effect on the performance of the plant (as measured by COD removal).

There is evidence that some species of micro-organism are much more tolerant to chromium (VI) than others. It has been reported that a strain of Pseudomonas aeruginosa was able to grow in the presence of 428 mg Cr (VI)/l (as potassium chromate), and another Pseudomonas species was
tolerant to 5,356 mg Cr (VI)/l (as potassium chromate). Similarly species of Athrobacter and Agrobacter could tolerate chromium (VI) concentrations up to 400 mg Cr (VI)/l and 100 mg Cr (VI)/l (as potassium dichromate) respectively. Gram positive bacteria are generally more
resistant to chromium (VI) than gram negative bacteria (Coleman, 1988).

Miranda and Castillo (1998) investigated the resistance of 172 motile Aeromonas sp. (associated with sewage treatment processes) isolated from Chilean raw drinking water supplies, irrigation waters and runoff waters receiving sewage to chromium (VI) (source not stated). In the
experiment, a Minimal Inhibitory Concentration (MIC) was determined using a growth media containing yeast extract, peptone and agar. The chromium (VI) concentration tested ranged between 5-700 mg/l and MICs in the range 20-300 mg/l were determined. Around 2-5% of the strains tested wereconsidered by the authors to be resistant to chromium (VI) (MIC>100 mg/l), and 50% of the strains tested had MICs in the range 40-80 mg/l.

Ross et al. (1981) looked at the effect of chromium (VI) (as potassium dichromate) and chromium (III) (as chromic chloride) on the growth of a mixed bacterial population isolated from soil. Aqueous soil extracts were used in the experiment (pH 6.5) using both non-aerated and aerated cultures at 23-33oC for up to 48 hours. A difference in sensitivity between gram negativeand gram positive bacteria was found in the study. The growth of all gram negative bacteria was found to be almost completely inhibited by 10-11 mg Cr (VI)/l. A concentration of 1 mg Cr (VI)/l had no effect on most gram positive bacteria, whereas significant growth inhibition was seen with some gram negative bacteria at the same concentration. Analysis after
48 hours indicated that around 80% of the chromium present was still as chromium (VI). The experiments with chromium (III) showed that it was much less toxic to the bacteria, with higher growth rates than controls being seen with some species at a concentration of 10 mg Cr (III)/l and only a small amount of growth inhibition being seen at a concentration of 100 mg Cr (III)/l.

The results of the microorganism toxicity studies evaluated in the EU RAR are summarised below:

Species	Endpoint	Value (mg/L)	Reference
Activated sludge
Mixed	3-hour IC50	30a	Klecka and Landi (1985)
Bacteria
Bacillus subtilis	10-hour EC50	0.11	Ogawa et al. (1989)
Escherichia coli	24-hour EC50	3.5 0.42b	Gaur and Bhattacherjee (1991)
Photobacterium Phosphoreum	30-minute EC50	21 27 200c	Tarkpea et al. (1986) Krebs (1983) Krebs (1983)
Pseudomonas Fluorescens	7-day NOEC	0.11	Sloof and Canton (1983)
Vibrio harveyi	50-minute EC50	2.2	Thomulka and Lange (1997)
Protozoa
Chilomonas paramecium	19-25-hour NOEC	1.0	Cairns Jr. et al. (1978)
Colpidium campylum	24-hour IC50	2.8	Dive et al. (1990)
Microregma heterostoma	28-hour NOEC	0.21	Bringmann and Kuhn (1959)

Notes: all results are from tests with potassium dichromate except for:
 a - sodium dichromate and b – sodium chromate.

c - solutions were pH neutralised.

All concentrations as Cr.

Validity criteria fulfilled:

yes

Conclusions:

Based on results from activated sludge (mixed), protozoa and bacteria, the toxicity of Cr (VI) as represented by potassium dichromate, sodium dichromate and sodium chromate are well described with I/EC50 values ranging from 0.11 to 200 mg/L. NOEC values range from 0.11 to 1.0 mg/L (total Cr).

Executive summary:

Based on a review of existing data on the toxicity of Cr(VI) to microbes, a range of I/EC50 and NOEC values were presented. There is evidence that some species of micro-organism are much more tolerant to chromium (VI) than others. Gram positive bacteria are generally more resistant to chromium (VI) than gram negative bacteria.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

published 1981

GLP compliance:

not specified

Specific details on test material used for the study:

sodium dichromate dihydrate used

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions (0.5 to 5.0 g/L) of the test chemicals were prepared in deionized water prior to inhibition testing. When necessary, the stock solutions were adjusted to pH 7.5 +/- 0.5 by the addition of 1N H2SO4 or 1N NaOH as required. A series of reaction mixtures were prepared containing a defined concentration of synthetic sewage and activated sludge inoculum with varying concentrations of the test chemical. Test reaction mixtures were prepared by adding 16 mL of synthetic sewage stock solution and the desired amount of the test chemical to a 500 mL graduated cylinder. The mixture was diluted to a volume of 300 mL with deionized water. Activated sludge inoculum (200 mL) containing approximately 800 mg of suspended solids (dry weight) was then added and the contents of the cylinder (final volume of 500 mL) were transferred to a 1L bottle. The reaction mixture was aerated at a rate between 0.5 and 1.0 L/min to ensure complete mixing using a pasteur pipette aeration device.
- Controls: Control reaction mixtures were prepared at the beginning and end of the study in which the test chemical were omitted. In addition, reaction mixtures were also prepared with at least 3 concentrations of the reference substance (3,5-dichlorophenol) to insure the sensitivity of the activated sludge was not abnormal.
- Test concentration separation factor: not specified
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: no
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge was obtained from a local municipal wastewater treatment plant.
- Preparation of inoculum for exposure: The activated sludge was obtained on the day prior to the first day of the inhibition test. On return to the laboratory, the solids were allowed to settle and the waste liquor was discarded. The solids were then transferred into a 9 L laboratory-scale, semi-continuous activated sludge cylinder and diluted to approximately 8.5L with deionised water. The system was initially mixed by aerating at a rate of approximately 0.5L/min, and the concentration of suspended solids was determined by gravimetric analysis. The solids were then allowed to settle in the cylinder and the upper layer of waste liquor was discarded. The activated sludge was washed three times with appropriate volumes of deionised water. After washing, the system was adjusted to contain 4000 ± 400 mg/L of suspended solids (dry weight). The system was then aerated continuously at a rate of 0.5 L/min and incubated at ambient temperature (21°C). The system was supplemented daily with 50 mL of a synthetic sewage stock solution per liter of activated sludge. The synthetic sewage stock solution was similar to that described by the OECD with the exception that the level of K2HPO4 was increased to 28.0 g for increased buffering capacity. For a detailed composition of the synthetic sewage stock solution please refer to section "Any other information on materials and methods incl. tables". Fresh stock solution was prepared as required and stored for not more than two days at 5°C.
- Initial biomass concentration: Activated sludge inoculum (200 mL) containing approximately 800 mg of suspended solids (dry weight).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21°C

pH:

7.4-8.0

Nominal and measured concentrations:

nominal, accurate values not stated

Details on test conditions:

TEST SYSTEM
- Test vessel: 1L bottles
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 1L bottles filled with 500 mL test solution
- Aeration: Yes, at a rate between 0.5 and 1.0 L/min.
- Sludge concentration (weight of dry solids per volume): 1.6 g/L
- Nutrients provided for bacteria: synthetic sewage (composition of stock solution as described in OECD 209 and presented in section "Any other information on materials and methods incl. tables")

OTHER TEST CONDITIONS
- Adjustment of pH: to pH 7.5 +/- 0.5 (reference compound stock solution and activated sludge prior to test start)
- Details on termination of incubation: Following the 3-hour incubation period, a portion of a reaction mixture was transferred to a standard BOD dilution bottle. The rate of oxygen consumption in the sample was immediately analyzed using an oxygen electrode and an Ionanalyser. An additional magnetic stirrer was added to the BOD bottle to ensure proper mixing of the sample, since the captive stirrer supplied with the oxygen electrode did not provide adequate mixing. Output of the Ionanalyzer was connected to a chart recorder, and the change in dissolved oxygen concentration in the sample was recorded over a 5 minute period. The respiration rate of the sample, defined as mg of O2/L consumed per minute, was calculated from the slope of the recorder trace between approximately 6.0 mg O2/L and 2.0 mg O2/L. Following analysis of the oxygen consumption rate, the pH of the sample was determined.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The effect parameter measured was the respiration rate/oxygen consumption rate.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (99%); stock solution prepared by dissolving 0.5g of 3,5-dichlorophenol in 10 mL of 1N NaOH and diluting to 30mL with deionized water

Duration:

3 h

Dose descriptor:

IC50

Remarks:

Probit-Transformation Analysis

Effect conc.:

75.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95%-confidence intervals: 18.6 - 307.6 mg/L

Key result

Duration:

3 h

Dose descriptor:

IC50

Remarks:

Moving Average Analysis

Effect conc.:

58.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

IC50

Remarks:

Graphic Analysis

Effect conc.:

81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

3h-IC50: 12.2 mg/L

Reported statistics and error estimates:

Inhibition data were analyzed using Thompson's method of moving averages to estimate IC50 values. The moving average method is an empirical approach for estimating median effect doses, based on linear interpolation between moving averages of the experimental data. The procedure is similar to the graphical approach where IC50 values are obtained from semi-log plots (log-linear interpolation between the two concentration-response values which bracket the 50% inhibitory response).
The experimental data were also analyzed using a probit-transformation model similar to that described by Larson and Schaeffer. A nonlinear curve-fitting program (Procedure NLIN of SAS) was used to estimate IC50 values and associated 95% confidence intervals.

Validity criteria fulfilled:

yes

Conclusions:

An activated sludge respiration inhibition test according to OECD TG 209 was conducted with sodium dichromate. The test conditions are well documented, the validity criteria were met and the results can be considered as reliable for the assessment of the toxicity of the registered substance to microorganisms.
The test was conducted by incubation of samples of activated sludge with a nutrient source to several concentrations of the test material and a control. Beside a reference control was running to proof the viability of the microorganisms. Following incubation under standard test conditions for a period of 3 h the rate of oxygen consumption was measured in each sample. Based on the obtained results the most sensitive 3h IC50 of nominal 58.7 mg/L was determined by moving average analysis with 95% confidence intervals of 18.6 - 307.6 mg/L. Beside, a 3h IC50 of nominal 75.5 mg/L was determined by Probit transformation analysis and a 3h IC50 of nominal 81.0 mg/L was determined by graphic analysis.

Executive summary:

Within the present study the OECD Activated Sludge, Respiration Inhibition Test (OECD TG 209) was evaluated as a method for assessing the potential impact of chemicals on wastewater treatment systems. Reproducibility of the test method was examined with respect to variation in oxygen consumption rates measured for a series of reactions set under identical conditions, and the variability in IC₅₀ values estimated for three reference compounds (mercuric chloride, 3,5-dichlorophenol and phenol). Bottle-to-bottle variation in respiration rates measured for a series of reactions was on the order of 5-6%, while variability in IC₅₀ values for the three compounds was in the range of 12-22%. In addition, IC₅₀ values for each of the three compounds were similar in studies conducted using activated sludge from both municipal and industrial sources. Several statistical procedures were also evaluated for analyzing activated sludge inhibition data. The test method was subsequently used to examine the effects of a wide variety of heavy metals on activated sludge respiration rates, including sodium dichromate.

The test itself was conducted by incubation of samples of activated sludge with a nutrient source to several concentrations of the test material and a control. Beside a reference control was running to proof the viability of the microorganisms. Following incubation under standard test conditions for a period of 3 h the rate of oxygen consumption was measured in each sample. Based on the obtained results the most sensitive 3h IC50 of nominal 58.7 mg/L was determined by moving average analysis with 95% confidence intervals of 18.6 - 307.6 mg/L. Beside, a 3h IC50 of nominal 75.5 mg/L was determined by Probit transformation analysis and a 3h IC50 of nominal 81.0 mg/L was determined by graphic analysis.

Description of key information

1) Key_ Toxicity to microorganisms by summary of data from European Union Risk Assessment Report: chromium trioxide, sodium chromate, sodium dichromate, ammonium dichromate and potassium dichromate; 3rd. Priority List; Volume 53: range for I/EC50: 0.11 to 200 mg/L; range for NOEC: 0.11 to1.0 mg/L (total Cr); based on results from activated sludge (mixed), protozoa and bacteria tests with potassium dichromate, sodium dichromate and sodium chromate.

2) Key_Toxicity to microorganisms: 3h IC50: 58.7 mg/L (nominal, moving average analysis), 75.5 mg/L (nominal, Probit transformation analysis), 81.0 mg/L (nominal, graphic analysis) for activated sludge according to OECD TG 209 (predominately domestic, static, freshwater)

Key value for chemical safety assessment

EC50 for microorganisms:

0.11 mg/L

EC10 or NOEC for microorganisms:

0.11 mg/L

Additional information

1) Based on a review of existing data on the toxicity of Cr(VI) to microbes, a range of I/EC50 and NOEC values were presented (I/EC50: 0.11 to 200 mg/L; NOEC: 0.11 to 1.0 mg/L (total Cr)). There is evidence that some species of micro-organism are much more tolerant to chromium (VI) than others. Gram positive bacteria are generally more resistant to chromium (VI) than gram negative bacteria.

2) Within the present study the OECD Activated Sludge, Respiration Inhibition Test (OECD TG 209) was evaluated as a method for assessing the potential impact of chemicals on wastewater treatment systems. Several statistical procedures were evaluated for analyzing activated sludge inhibition data. The test method was subsequently used to examine the effects of a wide variety of heavy metals on activated sludge respiration rates, including sodium dichromate.

The test itself was conducted by incubation of samples of activated sludge with a nutrient source to several concentrations of the test material and a blank and reference control. Following incubation under standard test conditions for a period of 3 h the rate of oxygen consumption was measured in each sample. Based on the obtained results the most sensitive 3h IC50 of nominal 58.7 mg/L was determined by moving average analysis with 95% confidence intervals of 18.6 - 307.6 mg/L. Beside, a 3h IC50 of nominal 75.5 mg/L was determined by Probit transformation analysis and a 3h IC50 of nominal 81.0 mg/L was determined by graphic analysis.