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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:
2018-05-02 to 2018-08-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the lot/batch: 2018-11-17 (retest date)
- Purity test date: 2018-08-31 (certificate of analysis release date)
- Purity: 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: no data
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- Method: The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. In the two combined limit/range-finding tests, the preparation of the test item concentrations started by adding weighed amounts of test item directly in to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period, with a maximum of 9 minutes. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the exposure period of 3 hours. Thereafter, oxygen consumption was recorded for approximately 10 minutes.
- Based on the results of the combined limit range finding test, the final tests was performed with two stirring/exposure period procedures to determine the inhibition of the respiration rate of the test item and the degradation product of the test item.
For the determination of the inhibition of the respiration rate of the test item (full test A), the preparation of the test concentrations started by adding weighed amounts of test item directly in to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period of approximately 6 minutes.
- For the determination of the inhibition of the respiration rate of the degradation product of the test item (Full test B), the preparation of the test concentrations started by adding weighed amounts of test item directly in to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred at room temperature, for a period of three days to ensure full degradation of the test item.
- In the full tests, subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations.
- Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the exposure period of 30 minutes (Full test A) or 3 hours (Full test B). Thereafter, oxygen consumption was recorded for approximately 10 minutes.
- Controls: blank controls: test medium without test item and treated in the same way as the test item solutions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: predominantly domestic sewage treatment plant, "Waterschap Aa en Maas", 's-Hertogenbosch, The Netherlands.
- Preparation of inoculum for exposure: coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ~105°C to determine the amount of suspended solids
- Pretreatment: The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per L of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g/L of sludge
Test type:
static
Water media type:
freshwater
Limit test:
no
Remarks on exposure duration:
The influence of JNJ-39722280-AAA (4-Nitrobenzene-1-sulfonyl chloride) on the respiration rate of activated sludge was investigated after a contact time of 30 minutes and 3 hours.
Post exposure observation period:
After 30 minutes, or 3h of exposure, respectively, oxygen consumption was recorded for approximately 10 minutes.
Test temperature:
-In Full test A (30 mins of exposure to the test item) and B (3 hours of expsoure to the degradation products), the temperature was above the range prescribed by the study plan (20 ± 2°C) during the exposure phase, the temperature was 20-23°C. This did not impact the performance of the study.
pH:
* Full test A (30 mins of exposure to the test item)
pH (start): 7.5-7.7
pH (end): 7.9-8.1
* Full test B (3 hours of expsoure to the degradation products)
pH (start): 7.5-7.7
pH (end): 7.9-8.1
Dissolved oxygen:
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
Salinity:
not applicable
Nominal and measured concentrations:
Loading rates for the test substance:
- Two Combined limit/range-finding tests: 10, 100 and 1000 mg/L
-Final tests A (30 mins of exposure to the test item) and B (3 hours of expsoure to the degradation products): 3.2, 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- Aeration: After either 30-minutes (test A) or 3-hour (tets B) contact time, oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
- Nuber of replicates:test
* Combined limit/range- finding tests: The highest loading rate in triplicate, lower loading rates in one replicate, control in six replicates and an abiotic control in one replicate.
* Final tests A and B: 5 replicates per test group and 6 replicates for the control.
- Biomass loading rate: initial loading ca. 3.0 g dw/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system. Oxygen consumption was monitored for approximately 10 min after the 30-min (test A) and 3-h (test B) exposure period, respectively. Respiration rate was calculated from the measurements.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x3.2
- Range finding study test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: Test A
Key result
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% C.I.: 15-110 mg/L Test A
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: Test A
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
other: degradation product of the test item
Basis for effect:
inhibition of total respiration
Remarks on result:
other: Test B
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
347 mg/L
Nominal / measured:
nominal
Conc. based on:
other: degradation product of the test item
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% C.I.: 314-384 mg/L Test B
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
517 mg/L
Nominal / measured:
nominal
Conc. based on:
other: degradation product of the test item
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% C.I.: 449-596 mg/L Test B
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: no data
- Blank controls oxygen uptake rate: 32.98 mg O2/L.h (test A) and 29.72 mg O2/L.h (test B)
- Coefficient of variation of oxygen uptake rate in control replicates: 5 and 12%, respectively for full test A and B
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 value of 11 and 6 mg/L in full test A and B, receptively
- Other: This value is within the recommended range of 2-25 mg/L confirming suitability of the activated sludge used.
Reported statistics and error estimates:
- EC50 values for the reference item was calculated using 3-parameter logistic cumulative distribution function (CDF) using a non-linear regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
- For the test item (full test A), calculation of the ECx value was based on logit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
- For the degradation product of the test item (full test B), calculation of the ECx value was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
- An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller).
- Finally,all calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).
Validity criteria fulfilled:
yes
Remarks:
all criteria for validity of the test were met
Conclusions:
An activated sludge respiration inhibition test was carried out with activated sludge from a predominantly domestic sewage treatment plant according to OECD guideline 209 and under GLP conditions.
Based on the results of the two combined limit/range-finding studies two full test were performed. Full test A is performed to determine the inhibitory effect of the test item on the reparation rate of the activated sludge. The test item was added directly in to the test vessel and an exposure period of 30 minutes to ensure that there was no complete degradation of the test item. Full test B was performed to determine the inhibitory effect of the test item on the reparation rate of the activated sludge. The test item was added directly in to the test vessel and stirred for 3 days to ensure the full degradation of the test item. The exposure period was 3 hours. Full study A resulted in a 30-min EC50 > 1000 mg 4-Nitrobenzene-1-sulfonyl chloride/L. Under the conditions of this present test 4-Nitrobenzene-1-sulfonyl chloride was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 10 mg/L (NOEC). An additional activated sludge respiration inhibition test (full study B) was conduted on the degradation product of the test item. This test gave rise to a 3-hour EC50 of 517 mg/L (95% C.I. 449-596 mg/L) and a 3-hour NOEC of 320 mg/L. The results of the study can be considered reliable without restriction.

Description of key information

The study of Buitenweg (2018), investigating the toxicity of 4-Nitrobenzene-1-sulfonyl chloride to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The influence of JNJ-39722280-AAA (4-Nitrobenzene-1-sulfonyl chloride) on the respiration rate of activated sludge was investigated after a contact time of 30 minutes of the test substance and after 3 hours for the degradation product of the test substance. The first full test (A) resulted in a 30-min EC50 > 1000 mg/L and a 30-min EC10 of 40 mg/L (95% C.I. 15 -110 mg/L) the test substance. Under the conditions of this present test JNJ-39722280-AAA (4-Nitrobenzene-1-sulfonyl chloride) was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 10 mg/L (NOEC). An additional activated sludge respiration inhibition test (full test B) was conducted on the degradation product of the test item. This additional test gave rise to a 3-hour EC50 of 517 mg/L (95% C.I. 449-596 mg/L) and a 3-hour EC10 of 347 mg/L (95% C.I. 314 -384 mg/L). Under the conditions of this present test the degradation product of JNJ-39722280-AAA (4-Nitrobenzene-1-sulfonyl chloride) was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 320 mg/L (NOEC). Because the degradation product was shown to be less toxic than the test item, the 30-min EC10 of 40 mg/L of the test substance was used as key value for chemical safety assessment in a conservative approach. The results of the study can be considered reliable without restriction.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
40 mg/L

Additional information