Thiophanate-methyl

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-20 to 2012-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test mixtures containing water, synthetic wastewater and the test item were prepared to obtain different nominal concentrations of the test item. All test item concentrations, 1000, 100 and 10 mg/L, were weighed directly into the test vessels. The test item concentrations 1000 and 100 mg/L were performed with a final volume of 500 mL whereas 10 mg/L test item concentration was performed with a final volume of 1000 mL.

- Controls: The abiotic control was prepared with the highest test item concentration, water and synthetic wastewater. The nitrification controls were prepared with a concentration of 11.6 mg ATU/L. These controls were performed for each concentration of the test assay and for the references assay.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Origin: municipal wastewater treatment plant of Pforzheim, Germany
- Preparation of inoculum for exposure: The sludge was used on the day of collection. It was settled for about 10 minutes and the upper layer with finer solids was decanted. Before starting the test, it was washed three times with chlorine free tap water by centrifugation (10 minutes at 1000 rpm). After centrifuging the supernatant was decanted and discarded and the sludge was resuspended in chlorine free tap water. This procedure was repeated twice. The mixed liquor suspended solids (MLSS) were adjusted to a concentration of 3.0 g/L (± 10 %). The activated sludge was continuously aerated at the test temperature, the solids must not settle down.
- Initial biomass concentration: 1.5 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.9 - 23.1 °C

pH:

7.04 - 7.62

Nominal and measured concentrations:

Nominal conc: 10, 100 and 1000 mg/L
No measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Material, size, fill volume: glass, 1 L, 1000 and 500 mL
- Aeration: yes
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- No. of vessels per abiotic control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 1.5 g/L dry matter
- Nutrients provided for bacteria: synthetic sewage
- Nitrification inhibitor used: N-allylthiourea

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

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous darkness

EFFECT PARAMETERS MEASURED: respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes
- 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 (DCP)

Key result

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

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

The total oxygen uptake rate was determined for all treatment groups but the inhibitory effects were only discussed for the highest test concentration of 1000 mg/L.
After 30 min, an inhibition effect at 1000 mg/L of -5.9 % (mean) was determined. After 3 h, an inhibitory effect of -1.6 % (mean) was determined. This effect was statistically not significant (p-value was with 0.6884 determined to be above 0.05).
For the heterotrophic oxygen uptake, after 30 min an mean inhibitory effect of -14.0 % was determined. After 3 h, an inhibitory effect of 3.2 % (mean) was determined. This effect was statistically not significant (p-value was with 0.2513 determined to be above 0.05).
The inhibitory effects of nitrifiers were determined to be 20.8 % after 30 min and -18.9 after 3 h. This effect was statistically not significant (p-value was with 0.8804 determined to be above 0.05).

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was within the recommended range of 2 to 25 mg/L. For the total oxygen uptake, the EC50 was determined to be 11.85 mg/L after 30 min and 8.24 mg/L after 3 h. For the heterotrophic oxygen uptake, the EC50 was determined to be 18.42 mg/L after 30 min and 12.33 mg/L after 3 h. For the oxygen consumption due to nitrification, the EC50 was determined after 30 min to be 0.87 mg/L and 1.33 mg/L after 3 h.

Table 1: Results of the range-finding test, specific respiration rates

	Specific respiration rates [mg O2*/(g x h)]
Test assay [mg/L]	Total		Heterotrophic		Due to nitrification
	30 min	3h	30 min	3h	30 min		3 h
Control 1	28.47	38.65	21.63	30.94	7.30*	8.26*
Control 2	31.95	36.25	24.97	30.01
Control 3	34.05	38.51	25.97	27.67
Mean	31.49	37.80	24.19	29.54	7.30	8.26
Std dev.	2.82	1.35	2.27	1.68	2.82	1.35
CV [%]	9	4	9	6	39	16
10 mg/L	29.37	33.07	21.79	23.95	7.58	9.12
100 mg/L	31.55	31.90	27.39	32.19	4.16	-0.29
1000 mg/L	3225	37.43	27.15	26.86	5.78*	9.82*
1000 mg/L	33.68	40.03	27.75	29.41
1000 mg/L	34.12	37.75	27.80	29.41
Mean	33.35	38.40	27.57	28.58	5.78	9.82
Std dev	0.98	1.42	0.36	1.49	0.98	1.42
DCP 2 mg/L	27.09	29.31	25.31	25.36	1.78	3.95
DCP 5 mg/L	26.23	27.06	25.70	26.05	0.53	1.01
DCP 10 mg/L	19.15	20.11	20.47	20.85	-1.32	-0.74
DCP 25 mg/L	6.77	5.11	7.38	4.47	-0.61	0.64
Abiotic control 1	0.03	0.35	-	-	-	-
Abiotic control 2	0.03	-0.15	-	-	-	-
Abiotic control 3	0.07	-0.05	-	-	-	-
Mean	0.04	0.05
Std.dev.	0.02	0.26

* the nitrification values were determined by calculating the difference between the mean of the total specific respiration rate and the mean of the heterotrophic respiration rate

 

Table 2: Results of the range-finding test, inhibition

	Inhibition [%]
Test assay [mg/L]	Total		Heterotrophic		Nitrifier*
	30 min	3h	30 min	3h	30 min	3h
10 mg/L	67	12.5	9.9	18.9	-3.8^1)	-10.4^1)
100 mg/L	-0.2^1)	15.6	-13.2^1)	-9.0D	43.0	103.5
1000 mg/L (mean)	-5.9^1)	-1.6^1)	-14.0^1)	3.2	20.8	-18.9^1)
Standard deviation	3.1	3.8	1.5	5.1	5.0	18.1
DCP 2 mg/L	14.0	22.5	-4.6D	14.2	75.6	52.2
DCP 5 mg/L	16.7	28.4	-6.2^1>	11.8	92.7	87.8
DCP 10 mg/L	39.2	46.8	15.4	29.4	118.1	109.0
DCP 25 mg/L	78.5	86.5	69.5	84.9	108.4	92.3
Abiotic control (mean)	99.9	99.9	-	-	-	-
Standard deviation	0.1	0.7	-	-	-	-

- no value

* the nitrification values were determined by calculating the difference between the mean of the total specific respiration rate and the mean of the heterotrophic respiration rate

¹⁾ negative value mean stimulating effect

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on microorganisms of an aerobic microbial treatment plant was tested with the activated sludge respiration inhibition test.
The test item had no toxic effects on the oxygen uptake at the highest concentration tested (1000 mg/L). Hence, the EC50 of the test item is > 1000 mg/L and the NOEC is 1000 mg/L.

Executive summary:

Toxicity testing of the test item to Microorganisms was done in an Activated Sludge Respiration Inhibition Test according to OECD 209. The respiration rate of an activated sludge, fed with a standard amount of synthetic wastewater feed, was measured for 30 minutes and 3 hours after application of the test item. The inhibitory effects of the test item in particular concentrations were expressed as a percentage of the mean respiration rate. The sludge was adjusted to a content of 1.5 g/L dry matter and was exposed to the test item under continuous aeration. After stopping the aeration, the O₂ consumption was measured for approx. 5 minutes. The slope of the O₂ consumption straight line is an indication for toxic effects on respiration activity of microorganisms. A range-finding test with concentrations from 10, 100 and 1000 mg/L in a limit test design was performed. A control consisting of three replicate was also included. Four concentrations of 3,5-dichlorophenol as toxic reference item were also tested to demonstrate the sensitivity of the test system. To test the differences between total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrification, two test assays were performed, one with and one without allylthiourea (ATU) respectively. All validity criteria were met. The total oxygen uptake rate was determined for all treatment groups but the inhibitory effects were only discussed for the highest test concentration of 1000 mg/L. The EC₅₀ of 3,5-dichlorophenol was within the recommended range of 2 to 25 mg/L. For the total oxygen uptake, the EC₅₀ was determined to be 11.85 mg/L after 30 min and 8.24 mg/L after 3 h. For the heterotrophic oxygen uptake, the EC₅₀ was determined to be 18.42 mg/L after 30 min and 12.33 mg/L after 3 h. For the oxygen consumption due to nitrification, the EC₅₀ was determined after 30 min to be 0.87 mg/L and 1.33 mg/L after 3h. The control oxygen uptake rates for total oxygen uptake were 31.49 and 37.80 mg 02/ (g x h) dry matter after 30 min and 3 h, respectively. The coefficients of variation of the control for total oxygen uptake were 9 % and 4 % after 30 min and 3 h, respectively. The coefficient of variation of the control with ATU was determined to be 9 % after 30 min and 6 % after 3 h. Both controls did not exceed 30 %. There was not less than 20 mg oxygen uptake per g activated sludge (dry weight of suspended solids) in 1 hour. The test item had no toxic effects on the oxygen uptake at the highest concentration tested (1000 mg/L). Hence, the EC₅₀ of the test item is > 1000 mg/L and the NOEC is 1000 mg/L.

Description of key information

The effect of the test item on microorganisms of an aerobic microbial treatment plant was tested with the activated sludge respiration inhibition test. The test item had no toxic effects on the oxygen uptake at the highest concentration tested (1000 mg/L). Hence, the EC₅₀ of the test item is > 1000 mg/L and the NOEC is 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Toxicity testing of the test item to Microorganisms was done in an Activated Sludge Respiration Inhibition Test according to OECD 209. The respiration rate of an activated sludge, fed with a standard amount of synthetic wastewater feed, was measured for 30 minutes and 3 hours after application of the test item. The inhibitory effects of the test item in particular concentrations were expressed as a percentage of the mean respiration rate. The sludge was adjusted to a content of 1.5 g/L dry matter and was exposed to the test item under continuous aeration. After stopping the aeration, the O₂ consumption was measured for approx. 5 minutes. The slope of the O₂ consumption straight line is an indication for toxic effects on respiration activity of microorganisms. A range-finding test with concentrations from 10, 100 and 1000 mg/L in a limit test design was performed. A control consisting of three replicate was also included. Four concentrations of 3,5-dichlorophenol as toxic reference item were also tested to demonstrate the sensitivity of the test system. To test the differences between total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrification, two test assays were performed, one with and one without allylthiourea (ATU) respectively. All validity criteria were met. The total oxygen uptake rate was determined for all treatment groups but the inhibitory effects were only discussed for the highest test concentration of 1000 mg/L. The EC₅₀ of 3,5-dichlorophenol was within the recommended range of 2 to 25 mg/L. For the total oxygen uptake, the EC₅₀ was determined to be 11.85 mg/L after 30 min and 8.24 mg/L after 3 h. For the heterotrophic oxygen uptake, the EC₅₀ was determined to be 18.42 mg/L after 30 min and 12.33 mg/L after 3 h. For the oxygen consumption due to nitrification, the EC₅₀ was determined after 30 min to be 0.87 mg/L and 1.33 mg/L after 3h. The control oxygen uptake rates for total oxygen uptake were 31.49 and 37.80 mg 02/ (g x h) dry matter after 30 min and 3 h, respectively. The coefficients of variation of the control for total oxygen uptake were 9 % and 4 % after 30 min and 3 h, respectively. The coefficient of variation of the control with ATU was determined to be 9 % after 30 min and 6 % after 3 h. Both controls did not exceed 30 %. There was not less than 20 mg oxygen uptake per g activated sludge (dry weight of suspended solids) in 1 hour.

The test item had no toxic effects on the oxygen uptake at the highest concentration tested (1000 mg/L). Hence, the EC₅₀ of the test item is > 1000 mg/L and the NOEC is 1000 mg/L.