CJ309

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From December 13, 2016 to May 09, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guideline and in accordance with GLP

Justification for type of information:

Demonstrates that the hypothesis is supported by referring to a data set: all claims must be supported by data.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

Deionised water instead of distilled water used for preparation of stock solution; preparation of the inoculum: higher sludge weight after the washing procedure (sludge concentration in the test according to the guidelines); 4 instead of 5 test conc

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

Formulation:
Just before the start of the experiment a concentrated stock solution (3200 mg/L) was prepared by dissolving test item in deionized water using ultrasonic bath.
The test solutions used in the test were freshly prepared by dilution of the stock solution by mechanical dispersion at the beginning of the experiment, in the testing laboratory.
The pH of the test solutions was checked but not adjusted because they were in the range of 7-8 at the start of the experiment.

Vehicle:

not specified

Details on test solutions:

Controls:
Abiotic Control (A)
In parallel to the study, abiotic control flasks (A1, A2, A3, A4 and A5) were tested without inoculum, but with the highest concentration of the test item (1000 mg/L nominal concentration), under identical test conditions.
Untreated Control (B)
1-1 member of five replicates (B1, B2, B3, B4 and B5) of the untreated control group (deionised water, synthetic sewage and inoculum, without addition of the test item) was tested in parallel with all of the test series.

In parallel with the test item, the reference item 3,5-Dichlorophenol (REF1, REF2, REF3, REF4) was tested (at nominal test concentrations of 1.0; 3.2; 10 and 32 mg/L) under identical test conditions.
A tock solution of 3,5-Dichlorophenol was prepared according to OECD209 (1.0g of 3,5-Dichlorophenol dissolved in 1000ml of water). Warm deionised water and ultra-sonication was used to help the dissolution of the reference substance and the volume of the solution was made up to 1.0L with deionised water, then cooled to room temperature. The pH of the solution was checked and adjusted with NaOH solution (2mol/L) to pH7.39.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The activated sludge was supplied from the sewage plant for domestic sewage in Veszprem, Hungry. It was supplied two days before the start of the experiment. During holding prior to use the sludge was fed daily with 50mL synthetic sewage per litre and kept aerated at 20±2℃ until use.
The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH7.59. The activated sludge was used directly after conditioning.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.5~21.8 °C

pH:

7.36~8.31

Nominal and measured concentrations:

The test was performed at three concentrations of the test item: 10 (TI11, TI12, TI13, TI14, TI15), 100 (TI21, TI22, TI23, TI24, TI24) and 1000mg/L (TI31, TI32, TI33, TI34, TI35) were used.

Details on test conditions:

Type and size: Appropriate glass breakers for 500 mL volume and BOD bottles with 300 mL volume.
Aetarion: With compressed air (about 1 L/min).

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.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

In comparison to the inoculum controls the inhibition of the respiration rate in the case of the activated sludge was between 0.67% and 8.22% in the examined range of 10-1000mg/L of CJ302.
The average specific respiration rate (Rs) of the untreated (Blank) controls (B1-B5) was 23.60 mg O2/L/h/g solid.
The variation coefficient of oxygen uptake rate in above untreated replicates was 6.74%.

Results with reference substance (positive control):

The following nominal concentrations of the reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as in the case of the test item: 1.0, 3.2, 10.0 and 32.0 mg/L.

In comparison to the controls the inhibition of the respiration rate of the activated sludge was 67.49% at the highest nominal concentration of 320. Mg/L. At the nominal concentrations of 1.0, 3.2 and 10.0 mg/L 9.38%, 14.53% and 13.53% inhibition of the respiration rate was calculated respectively.

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 24.94 mg/L with 95% confidence limits of 21.57-28.31mg/L.

Table 2. Influence of test item on oxygen consumption of activated sludge

Test group		Conc. Of test item in test mix (mg/L)	Total oxygen consumption rate (RT) (mg O2/L/h)	Specific respiration rate (RS) (mg O2/L/h/g solid))	Inhib. (%)	pH values^#		Temperature (℃) (measured in the Lab)*	Variation coefficient of O2 consumption rate (%)
ID	Name					start	end
A1	Abiotic	1000	-1.08	-	-	7.50	7.77	19.5/21.8	-
A2		1000	-1.32	-	-	7.47	7.74		-
A3		1000	-0.83	-	-	7.48	7.73		-
A4		1000	-1.12	-	-	7.49	7.74		-
A5		1000	-1.20	-	-	7.51	7.75		-
B1	Blank	0.00	37.20	24.80	-	7.36	8.05	19.5/21.8	6.74
B2		0.00	38.14	25.43	-	7.55	8.17
B3		0.00	33.84	22.56	-	7.75	8.15
B4		0.00	35.54	23.69	-	7.74	8.26
B5		0.00	32.31	21.54	-	7.69	8.34
REF1	Ref. item	1.00	30.98	20.65	9.38	7.50	8.15	19.5/21.8	-
REF2		3.20	29.15	19.44	14.53	7.52	8.16		-
REF3		10.00	29.51	19.67	13.53	7.53	8.18		-
REF4		32.00	10.4	6.93	67.49	7.53	8.19		-
TI1	Test item	10	34.06	22.71	0.67	7.79	8.11	19.5/21.8	-
TI2		100	32.79	21.86	4.25	7.73	8.14		-
TI3		1000	31.39	20.92	8.22	7.68	8.31		-

#: Individual pH values of the BOD flasks, except TI1-3, where the average are reported

*: Minimum / Maximum temperature (℃) measured in the Lab, during the experiment

Validity criteria fulfilled:

yes

Conclusions:

Activated sludge respiration inhibition test (carbon and ammonium oxidation) was carried out with CJ302 to evaluate the influence of the test item on the activity of activated sludge microorganisms by measuring the respiration rate under defived conditions.

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of CJ302 was the following:
 
The 3-hour EC50>1000mg/L
 
Based on the results of this study, the test item CJ302 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.

Executive summary:

A laboratory test was carried out with CJ302 to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

 

The test concentrations (10, 100 and 1000 mg/L) were chosen to permit the determination of the approximate EC50.

 

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

 

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of CJ302 was the following:

 

The 3-hour EC50>1000mg/L

 

Based on the results of this study, the test item CJ302 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.

Description of key information

Based on the read across result, a laboratory test was carried out with CJ302 to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

 

The test concentrations (10, 100 and 1000 mg/L) were chosen to permit the determination of the approximate EC50.

 

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

 

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of CJ302 was the following:

 

The 3-hour EC50>1000mg/L

 

The test item CJ302 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary. Hence, the in vitro mammalian cell gene mutation test was negative (OECD TG476).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

Activated sludge respiration inhibition test (carbon and ammonium oxidation) was carried out with CJ302 to evaluate the influence of the test item on the activity of activated sludge microorganisms by measuring the respiration rate under defived conditions.

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of CJ302 was the following:

 

The 3-hour EC50>1000mg/L

 

Based on the read across result, CJ302 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. Hence,the in vitro mammalian cell gene mutation test was negative (OECD TG476).