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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08.08.1988 - 10.08.1988 (experimental phase)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
The study was properly conducted, in accordance with an established guideline and to GLP, and the validity criteria were fulfilled. However the report omits some details of the characterisation of the test substance and of the test procedure.
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
yes
Remarks:
The suspended sludge solids concentration in the incubation mixtures (12.5 mL inoculum @ 6.0 g dry solids/250 mL total test vessel contents) was 300 mg/L, which is lower than the 1500 and 1600 mg/L stipulated in ISO 8192 and OECD 209, respectively.
Principles of method if other than guideline:
ISO 8192 (1986) method is equivalent to OECD Guideline 209 (1984) and ETAD method 103.
GLP compliance:
yes
Analytical monitoring:
not required
Details on sampling:
Not specified.
Vehicle:
no
Details on test solutions:
'Chromosal B' was weighed directly into the appropriate test vessels.
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: activated sludge maintained in a 3 L laboratory unit (OECD)
- Preparation of inoculum for exposure: not stated
- Pretreatment: not stated
- Initial biomass concentration: 6.0 g dry solids/L

The suspended sludge solids concentration in the incubation mixtures (12.5 mL inoculum @ 6.0 g dry solids/250 mL total test vessel contents) was 300 mg/L, which is lower than the 1500 and 1600 mg/L stipulated in ISO 8192 and OECD 209, respectively. The inoculum was obtained from a laboratory-scale STP unit that is likely to have been operated with a feed of OECD synthetic sewage, the same substrate as was provided in the respiration inhibition test. The specific respiration rate of the inoculum was high and it is likely that a reduction of the sludge solids density was necessary to be able to follow the decline of DO concentrations reliably in each of the test vessels. Performing the study with a lowered inoculum density would have had the effect of increasing the test substance:biomass ratio and making the test conditions more conservative; i.e. toxicity more likely to be over- than under-estimated.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not relevant, hardness regulated by the composition of the OECD synthetic sewage.
Test temperature:
Temperatures at measurement in definitive test (end of 3 h incubation): 23.0 to 25.2 degrees C.
pH:
pH at measurement in definitive test (end of 3 h incubation): 6.1 to 8.1.
Dissolved oxygen:
Not measured during the 3 h incubation.
During the definitive test, respiration rates were based on measurements of dissolved oxygen (DO) concentrations at the end of the 3 h incubation that were initially between 3.20 and 9.10 mg O2/L. It may be assumed that DO concentrations were at least this high during the incubation.
Salinity:
Not relevant.
Nominal and measured concentrations:
'Chromosal B' was applied in the definitive test at nominal concentrations of: 1000, 1800, 3200, 5600 and 10000 mg/L. The test substance was also applied at 10000 mg/L in an abiotic control (no inoculum added) to check for oxygen uptake by autooxidation. Exposure concentrations were not measured, in compliance with the requirements of the ISO and OECD test guidelines.
Details on test conditions:
TEST SYSTEM
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): two
- No. of vessels per abiotic control (replicates): one
- Biomass loading rate: 300 mg dry suspended solids/L in all vessels except the abiotic control


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : determination of the rate of decline of dissolved oxygen concentrations measured in the contents of each vessel after 3-h incubation.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: x ca. 1.8 (1000, 1800, 3200, 5600 and 10000 mg/L) in definitive test.
- Range finding study
- Test concentrations: 100, 1000 and 10000 mg/L
- Results used to determine the conditions for the definitive study: Inhibition of respiration 0, 0 and 44% at 100, 1000 and 10000 mg/L, respectively, in the range-finder.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The report claims that 'Chromosal B' was "water-soluble" under the conditions of the test.
Results with reference substance (positive control):
Inhibition in range-finder: 8% and 74% respiration inhibition at 1.0 and 20.0 mg 3,5-DCP/L, respectively;
Inhibition in definitive test: 0% and 65% respiration inhibition at 1.0 and 20.0 mg 3,5-DCP/L, respectively.

The validity of the test was confirmed by graphical evaluation of the response obtained with the reference substance. (Guideline stipulates a 3-h EC50 of between 5 and 30 mg/L for 3,5-DCP; the endpoint for 3,5-DCP is presumed to fall in this range although the result of the graphical evaluation is not stated).
Reported statistics and error estimates:
Not relevant.

The results of the definitive test are presented in the table below:

‘Chromosal B’ nominal concentration (mg/L)

respiration rate,
mg O2/L*h

respiration inhibition (%) relative to control

0 (inoculated blank control at beginning)

17

n.a.

0 (inoculated blank control at end)

1000

19

0

1800

22

0

3200

21

0

5600

18

0

10000

19

0

10000 (un-inoculated abiotic control)

0

n.a.

1.0 mg 3,5-DCP/L

18

0

20.0 mg 3,5-DCP/L

6

65

Individual rates data for the two control replicates are not provided in the report but can be calculated from raw data, as {(initial DO - final DO)/measurement interval in minutes}x 60 mg O2/L*h. Thus control at beginning: {(4.20-3.60)/2.0} x 60 = 18.0 mg O2/L*h, control at end: {(3.20 -2.40)/3.0} x 60 = 16.0 mg O2/L*h. Mean control rate = 22.9 mg O2/L*h. Maximum deviation between duplicate controls = (18.0-16.0)/16.0 x 100 = 12.5%.

No oxygen uptake occurred in the abiotic control which demonstrates that 'Chromosal B' did not undergo auto-oxidation under the conditions of the test.

Validity criteria fulfilled:
yes
Remarks:
#1: The EC50 for 3,5-DCP is not stated but was between 5 and 30 mg/L. #2: Divergence between replicate control respiration rates did not exceed 15%.
Conclusions:
The respiration of activated sludge was unaffected by 3-h exposure to 'Chromosal B' (chromium hydroxide sulphate) at nominal concentrations of between 1000 and 10000 mg/L. Based on nominal concentrations, the 3-hour EC50 was >10000 mg 'Chromosal B'/L.
Executive summary:

The effect of 'Chromosal B' (chromium hydroxide sulphate) on aerobic biological sewage treatment processes was assessed according to ISO Standard 8192 (equivalent to OECD Guideline 209 and ETAD method 103), by determining inhibition of respiration of the mixed community of microorganisms present in a sample of activated sludge. Activated sludge obtained from a laboratory unit was exposed over a period of three hours to 'Chromosal B' weighed directly into the appropriate test vessels at nominal concentrations of 0 (control), 1000, 1800, 3200, 5600 and 10000 mg/L. A pair of test vessels was allocated to the control and the test substance treatments were run singly. There was also an uninoculated abiotic control containing 10000 mg 'Chromosal B'/L and the reference inhibitor 3,5 -dichlorophenol was run at concentrations of 1.0 and 20.0 mg/L. No respiration inhibition, relative to the mean control rate, occurred in any of the 'Chromosal B' treatments and the 3 -h EC50 was therefore >10000 mg 'Chromosal B'/L, the highest concentration tested.

Description of key information

Key_ISO Standard 8192 (OECD 209), GLP, potassium chromium hydroxide sulpfate (as a realtive insoluble Cr(III) salt), nominal concentrations of 0 (control), 1000, 1800, 3200, 5600 and 10000 mg/L,

3 -h EC50 was therefore >10000 mg 'Chromosal B'/L (> 110 mg Cr/L), the highest concentration tested.

Key value for chemical safety assessment

EC50 for microorganisms:
110 mg/L

Additional information

The available toxicity data for chromium(III) has been derived mainly using the water soluble forms (chromic chloride, chromic nitrate and chromium potassium sulphate). In the environment, chromium(VI) is likely to be reduced to chromium(III) species that are much less soluble, and hence bioavailable, than the forms of chromium(III) used in these tests. When the more insoluble forms of chromium(III) (e.g. chromium hydroxide sulphate and dichromium trioxide) have been tested, they have generally shown no effects on aquatic organisms at concentrations up to their effective water solubility.

From the available toxicity data, it appears that chromium(III) is less toxic in hard water and saltwater than in soft water.

A concentration of 10 mg Cr(III)/L promoted growth in some species of bacteria, and 100 mg Cr(III)/L produced only a small amount of growth inhibition. The study did not provide a formal NOEC; it is proposed to use 10 mg Cr(III)/L as the PNEC for soluble chromium (III).

From: Appendix F: EU RAR (2005), Vol. 53: Table F.3 Summary of the ecotoxicological data for chromium (III) to bacteria

Micro-organism

Substance

Method

Endpoint
(mg Cr/L)

Reference

Activated sludge

Chromium hydroxide sulphate

ISO 8192-inhibition of oxygen consumption

3 hour NOEC > 3130

IUCLID, 1999

Azobacter vinelandii (soil bacterium)

Chromic chloride

Growth inhibition

over 4 days

LOEC/NOEC ~ 0.26

Ueda et al., 1988

Fusarium oxysporum (soil fungus)

Chromic chloride

Growth inhibition

over 27 hours

NOEC > 6.5

Ueda et al., 1988

Pseudomonas fluorescens

Dichromium trioxide

24h-NOEC >6,840

IUCLID, 1999

Pseudomonas fluorescens

Chromium hydroxide sulphate

24h-NOEC >313

IUCLID, 1999

The effect of 'Chromosal B' (chromium hydroxide sulphate) on aerobic biological sewage treatment processes was assessed according to ISO Standard 8192 (equivalent to OECD Guideline 209 and ETAD method 103), by determining inhibition of respiration of the mixed community of microorganisms present in a sample of activated sludge. Activated sludge obtained from a laboratory unit was exposed over a period of three hours to 'Chromosal B' weighed directly into the appropriate test vessels at nominal concentrations of 0 (control), 1000, 1800, 3200, 5600 and 10000 mg/L. A pair of test vessels was allocated to the control and the test substance treatments were run singly. There was also an uninoculated abiotic control containing 10000 mg 'Chromosal B'/L and the reference inhibitor 3,5 -dichlorophenol was run at concentrations of 1.0 and 20.0 mg/L. No respiration inhibition, relative to the mean control rate, occurred in any of the 'Chromosal B' treatments and the 3 -h EC50 was therefore >10000 mg 'Chromosal B'/L, the highest concentration tested.

The chromium content (molecular weight) of chromium hydroxide sulphate will be taken into account to derive the 3-hour EC50 > 110 mg Cr/L.  This is consistent with the EU RAR data on toxicity to microorganisms.

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