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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:
supporting study
Study period:
16 November 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
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
A stock solution of 10 g/L was prepared by adding 5.0 g test substance to 500 ml of Milli-RO water (tap water purified by reverse osmosis and vortexted to accelerate dissolution and to ensure homogeneity. The pH of the clear and colourless stock was adjusted to 8.0 using 1M HCl. Volumes of the stock solution corresponding to the test concentration were then added to the test vessels.

- Controls:
Blank-control, nitrification control, abiotic control and the highest concentration with a nitrification inhibitor
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure:
The sludge was coarsely sieved (1 mm), 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 (3 g/L of sludge, as used for the test). The pH was 7.2 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:
no
Total exposure duration:
3 h
Test temperature:
19.1 - 21.0°C
pH:
Start (before addition of sludge): 7.5 - 7.6
End: 7.4 - 7.9
Nominal and measured concentrations:
Nominal concentrations: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass, open bottles/vessels
- Material, size, headspace, fill volume: Glass, with a capacity of 1000 mL and filled with 500 mL.
- Aeration: yes
- No. of vessels per concentration (replicates): highest concentration was tested in triplicate; lower concentrations only had one replicate
- No. of vessels blank-control (replicates): 2
- Loading rate: 1.5 g/L of sludge

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q)

Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Synthetic medium (=sewage feed)
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10x
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(TMAH pentahydrate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 503 mg/L
Nominal / measured:
nominal
Conc. based on:
other: TMAH
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: corrected for water content
Details on results:
- In the combined limit/range-finding test an average inhibition of the respiration rate of 3, 13 and 32% was observed at test concentrations of 10, 100 and 1000 mg/L, respectively. The inhibition observed at 1000 mg/L was statistically significant (Two Sample t-Test: α=0.05 Toxstat).

- No NOEC could be determined.

- There was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed no heterotrophic inhibition of the respiration rate.

- Based on the molecular weight of TMAH pentahydrate (181.2 g/mol) and TMAH (91.15 g/mol) it can be calculated that the EC50 value of > 1000 mg/L test material (TMAH pentahydrate) corresponds with > 503 mg/L TMAH.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was 8.9 mg/L
Validity criteria fulfilled:
yes
Conclusions:
After 3 hours exposure, TMAH pentahydrate significantly inhibited the respiration rate of waste water micro-organisms (activated sludge) at 1000 mg/L. However, the EC50 exceeded the highest test concentration of 1000 mg/L and no NOEC could be determined. - Based on the molecular weight of TMAH pentahydrate (181.2 g/mol) and TMAH (91.15 g/mol) it can be calculated that the EC50 value of > 1000 mg/L test material (TMAH pentahydrate) corresponds with > 503 mg/L TMAH.
Executive summary:

The toxicity of TMAH pentahydrate to microorganisms was determined in a 3 h activated sludge respiration inhibition study carried out according to OECD guideline No. 209 and GLP principles.

In a combined limit/range-finding test, microorganisms in activated sludge were exposed to the test substance at concentrations of 10, 100 and 1000 mg/L under static conditions. AFter 3 hours exposure, TMAH pentahydrate significantly inhibited the respiration rate of waste water micro-organisms (activated sludge) at 1000 mg/L. However, the EC50 exceeded the highest test concentration of 1000 mg/L and no NOEC could be determined. Based on the molecular weight of TMAH pentahydrate (181.2 g/mol) and TMAH (91.15 g/mol) it was calculated that the EC50 value of > 1000 mg/L test material (TMAH pentahydrate) corresponds with > 503 mg/L TMAH.

All criteria for acceptability of the test were met and this study is considered to be valid. The present toxicity study is considered reliable without restrictions according to the OECD guideline No. 209.

Description of key information

MTBAC was concluded to be not toxic to the inoculum in a ready biodegradability test carried out according to OECD301B and GLP (Soeter, 2015) at a concentration of 18 mg/L, as in the toxicity control more than 25% biodegradation occurred within 14 days (40%, based on ThCO2).

In accordance with IR/CSA guidance R.10 (May 2008), paragraph 10.4.2, this concentration can be used as a NOEC value for the calculation of a PNEC for STP microorganisms. The conclusion on the toxicity of MTBAC to STP micro-organisms is supported by a study with the structurally similar substance TMAH pentahydrate (see read-across justification in IUCLID section 13).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
18 mg/L

Additional information

The conclusion on the toxicity of MTBAC to STP micro-organisms is supported by a study with the structurally similar substance TMAH pentahydrate (see read-across justification in IUCLID section 13). The toxicity of TMAH pentahydrate to microorganisms was determined in a 3 h activated sludge respiration inhibition study carried out according to OECD guideline No. 209 and GLP principles (Desmares-Koopmans, 2013). The study was considered valid and reliable.

In a combined limit/range-finding test, microorganisms in activated sludge were exposed to TMAH pentahydrate at concentrations of 10, 100 and 1000 mg/L under static conditions. The substance significantly inhibited the respiration rate of waste water (activated sludge) at 1000 mg/L. However, the EC50 exceeded the highest test concentration of 1000 mg/L and no NOEC could be determined. Based on the molecular weight of TMAH pentahydrate (181.2 g/mol) and TMAH (91.15 g/mol) it was calculated that the EC50 value of > 1000 mg/L test material (TMAOH pentahydrate) corresponds with > 503 mg/L TMAH.

Since the NOEC of MTBAC could be derived from the OECD301B study (Soeter, 2015), this value was considered for the CSA (worst case approach).