Tetrakis(phenylmethyl)thioperoxydi(carbothioamide)

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:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Remarks:

There were no deviations to the study plan.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Details on sampling:

Not necessary.

Vehicle:

no

Details on test solutions:

- The test item TBzTD (Tetrabenzyl thiuram disulfide) is not soluble in water. Therefore, the test item was directly dosed into each test flask and tap water was added. For complete suspension of the test item, this mixture was stirred for about 24 hours at 22°C in the dark / diffuse light before adding the inoculum. The test flasks were tightly closed to avoid water loss by evaporation.
- Nominal 10, 32, 100, 320 and 1000 mg test item/L

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Species / Origin: Activated sludge, micro organisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Darmstadt, Germany.
Conditioning: The activated sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. The latter procedure was repeated three times. The washed sludge was re-suspended in tap water and sewage feed (50 mL per L, see 6.6) was added. The sludge was kept at room temperature under continuous aeration until use overnight.Immediately before use, the dry weight of the activated sludge was determined, and diluted to 3.8 g/L with tap water. The pH of the activated sludge was 7.5 and therefore no adjustment was necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation period.

Hardness:

Not relevant: synthetic sewage feed according to GL 209

Test temperature:

Surrounding Type: Controlled environment room
Temperature: 22 °C during dissolution phase (24 hours)
19 - 21 °C during pre-incubation (3 hours)
18 - 20 °C during evaluation period

pH:

7.5

Dissolved oxygen:

The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentra-tions were at least 8.4 mg O2/L and thus >6.5 mg O2/L.

Salinity:

The test was performed in freshwater.

Nominal and measured concentrations:

Nominal 10, 32, 100, 320 and 1000 mg test item/L.

Details on test conditions:

- Type and Size: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume
- Identification: Each test unit was uniquely identified with the study number, treatment and replicate number.
- Preparation of Test Flasks: One test solution with a final volume of 500 mL was tested per treatment in a glass flask (>500 mL). Samples were prepared in about 15-minute intervals.The flasks were filled according to the scheme given in Table in "Any other information on materials and methods incl. tables.
- Synthetic Sewage: 16 g peptone
11 g meat extract
3 g urea
filled up to 1 litre0.7 g NaCl
with deionised water0.4 g CaCl2 · 2H2O
0.2 g MgSO4 · 7H2O
2.8 g K2HPO4
- Incubation Period: 3 hours
- Measurement of Respiration Rate: For the measurement of the respiration rate a well-mixed sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with an oxygen electrode, and was recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve, and were determined in the range between approximately 7.9 – 9.2 mg O2/L.
- Measurement of pH, Dissolved Oxygen and Water Temperature: The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all treatments. The water temperature was measured in one control medium at the start and the end of the incubation period.

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:

not specified

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Inhibition of Respiration Rate: The influence of TBzTD (Tetrabenzyl thiuram disulfide) on the respiration rate of activated sludge is presented in Table 2 and in Figure 1.The respiration rates of the activated sludge treated with the test item at concentrations between 10 mg/L and 1000 mg/L differed by less than 10% from control.Test item concentrations exceeding 1000 mg/L were not tested.

- 3-hour EC20 and 3-hour EC50: Based on measured inhibition rates, the 3-hour EC10, EC20 and EC50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 10% inhibition was noted after three hours incubation. No dose related response was found. The maximum deviation from the control in respiration rates was 5.7% at concentrations of 10 - 320 mg and 0% at 1000 mg TBzTD (Tetrabenzyl thiuram disulfide) per litre. Thus, the 3-hour EC10, EC20 and EC50 are clearly higher than 1000 mg/L under the present test conditions.The test according to OECD 209 guideline does not provide a threshold value for the determination of a NOEC. If a deviation of less = 15% between test item treated activated sludge and control is considered not be an effect, the NOEC of TBzTD (Tetrabenzyl thiuram disulfide) will be established above 1000 mg/L.

Results with reference substance (positive control):

- Inhibition of Respiration Rate: The following nominal concentrations of the positive control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 32, 10 and 3.2 mg/L. In comparison to the controls the respiration rate of the activated sludge was moderately inhibited by 20.0% at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg test item/L, the respiration rate was inhibited by 51.4 and 77.1%, respectively. The results are summarized in Table 2 and in Figure 1.

- 3-hour EC50 of 3,5-Dichlorophenol: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 10.5 mg/L. Due to mathematical reasons, no 95 % confidence limits could be determined.

Reported statistics and error estimates:

The inhibitory effect of the test item respectively of the refer-ence item at a particular concentration on the respiration rate were expressed as percentage of the mean value of the respiration rates of the two controls.
The 3-hour ECX including the 95 %-confidence limits of the test item and reference item were calculated by Probit analy-sis. The 3-hour EC50 of the test item could not be calculated due to the low toxicity of the test item to micro organisms under the tested conditions.

Influence of TBzTD (Tetrabenzyl thiuram disulfide) and the Reference Item3,5-Dichlorophenolon
Oxygen Consumption of Activated Sludge

Flask No.	Treatment	Concen­tration	Oxygen consumption	Inhibition	pH-values		Oxygen concentration [mg O2/L]
		[mg/L]	[mg O2/L min]	%	start*	end*	start*	end*
1	control	---	0.35	---	7.6	8.6	8.1	8.4
10	control	---	0.34	---	7.6	8.6	8.1	8.5
mean			0.35	---	---	---	---	---
% replicate deviation			2	---	---	---	---	---
2	3,5-DCP	32	0.08	77.1	7.6	8.5	7.8	9.2
3	3,5-DCP	10	0.17	51.4	7.6	8.6	8.0	8.9
4	3,5-DCP	3.2	0.28	20.0	7.6	8.6	8.0	8.8
5	Test item	1000	0.35	0	7.6	8.6	8.1	8.5
6	Test item	320	0.33	5.7	7.6	8.6	8.1	8.5
7	Test item	100	0.33	5.7	7.6	8.6	8.1	8.5
8	Test item	32	0.33	5.7	7.6	8.6	8.0	8.7
9	Test item	10	0.33	5.7	7.6	8.6	7.9	8.6

*: Start and end of 3-hour aeration
DCP:3,5-Dichlorophenol
Test item: TBzTD (Tetrabenzyl thiuram disulfide)

Validity criteria fulfilled:

yes

Remarks:

See conclusions

Conclusions:

No significant bacterial toxicity (inhibition of respiration of activated sludge) up to 1000 mg/L .
All the validity criteria were fullfiled:
- Inoculum Control: The respiration rates of the two controls did not differ by more than 15 %.
- Reference Item: The 3-hour EC50 of the reference item 3,5-Dichlorophenol was determined to be 10.5 mg/L and is thus in the range of 5 to 30 mg/L for the used activated sludge batch.
- Dissolved Oxygen: The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 8.4 mg O2/L and thus >6.5 mg O2/L.

Executive summary:

The purpose of this study was to determine the effect of the test item on bacteria according to the OECD guideline 209 “Activated Sludge, Respiration Inhibition Test”.

The effect is assessed by measuring the micro-organisms respiration rate on a sample of activated sludge during an incubation period of 3 hours.

The method was applied with respect to the following criteria:

The difference between the 2 controls was lower than 15 %;

EC₅₀of the reference item 3,5-dichlorophenol in the control test was beween 5 and 30 mg/l (10.5 mg/L);

Tetrabenzyl thiuram disulf ide did not show any effect on the respiration of activated sludge in the conditions of the test up to a concentration of 1000 mg/L.

Description of key information

Tetrabenzyl thiuram disulfide did not show any effect on the respiration of activated sludge in the conditions of the test up to a concentration of 1000 mg/L.

Additional information

The purpose of this study was to determine the effect of the test item on bacteria according to the OECD guideline 209 “Activated Sludge, Respiration Inhibition Test”. The effect is assessed by measuring the micro-organisms respiration rate on a sample of activated sludge during an incubation period of 3 hours.Tetrabenzyl thiuram disulfide did not show any effect on the respiration of activated sludge in the conditions of the test up to a concentration of 1000 mg/L.