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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:
On 8 June, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The batch of ebecryl® 140 radiation curing resins, an UVCB, was a clear colourless liquid and not completely soluble in test medium at the loading rates initially prepared. Preparation was as much as possible performed under dimmed light conditions. Therefore, 1 L test bottles were filled with 200 mL of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 h. Subsequently, 16 mL synthetic medium, 250 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (2.93 g/L of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
After the 3-hour contact time, the oxygen consumption was recorded for a period of 10 to 14 min. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture.
Test temperature:
Between 18.5 and 20.6°C
pH:
Before addition of sludge was between 7.3 and 7.4.
After the 3 h exposure period the pH was between 7.1 and 8.1.
Dissolved oxygen:
The respiration rate from each vessel, in mg O2/L/h, was calculated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/L.

Oxygen consumption:
Controls : 30 mg O2/L/h (mean value)
EBECRYL® 140 radiation curing resins at 10 mg/L : 33 mg O2/L/h
EBECRYL® 140 radiation curing resins at 100 mg/L : 31 mg O2/L/h
EBECRYL® 140 radiation curing resins at 1000 mg/L : 29, 36 and 28 mg O2/L/h
Nominal and measured concentrations:
Nominal loading rates: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: All glass
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 1 for 10 and 100 mg/L and 3 for 1000 mg/L (loading rates)
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1.5 g/L suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO)
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate from each vessel, in mg O2/L/h, was calculated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/L.

TEST CONCENTRATIONS
- Test concentrations: combined limit/range-finding test at 10, 100 and 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg test substance per litre (Two Sample t-Test : α=0.05 Toxstat).

There was no oxygen uptake from abiotic processes and the results at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (7.7 mg/L).
Reported statistics and error estimates:
ECx:
For the reference substance, the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.
For the test substance, no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > a loading rate of 1000 mg/L).

NOEC estimation:
The NOEC was based on statistical analysis of the data. Data obtained for the test concentrations were compared with those obtained for the control using TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.
Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, ebecryl 140 was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/L (NOEC). The EC50 exceeded a loading rate of 1000 mg/L.
Executive summary:

A study was conducted to determine the influence of di-TMPTTA on the respiration rate of activated sludge according to OECD Guideline 209, Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007), in compliance with GLP. A combined limit/range-finding test was conducted using loading rates of 0 (control), 10, 100 and 1,000 mg/L. The exposure duration was 3 h. No analytical dose verification was conducted. No statistically significant inhibition of the respiration rate of the sludge was recorded up to the highest loading rate of 1,000 mg/L. Further, there was no oxygen uptake from abiotic processes and the results at 1,000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. In conclusion, the test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1,000 mg/L (3 h NOEC). The 3 h EC50 exceeded a loading rate of 1,000 mg/L (Desmares-Koopmans, 2012).

Description of key information

Based on the study results, the test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1,000 mg/L (3 h NOEC, nominal). The 3 h EC50 exceeded a loading rate of 1,000 mg/L (nominal).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

A study was conducted to determine the influence of the test substance on the respiration rate of activated sludge according to OECD Guideline 209, Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007). A combined limit/range-finding test was conducted using loading rates of 0 (control), 10, 100 and 1,000 mg/L. The exposure duration was 3 h. No analytical dose verification was conducted. No statistically significant inhibition of the respiration rate of the sludge was recorded up to the highest loading rate of 1,000 mg/L. Further, there was no oxygen uptake from abiotic processes and the results at 1,000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. In conclusion, the test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1,000 mg/L (3 h NOEC). The 3 h EC50 exceeded a loading rate of 1,000 mg/L.