Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate

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:

24 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

- the respiration rate of the blank controls did not meet the minimum requirement. However, the high EC50 obtained for the test material and the good results obtained with the positive control indicate that this did not affect the validity of the test.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

yes

Remarks:

- the respiration rate of the blank controls did not meet the minimum requirement. However, the high EC50 obtained for the test material and the good results obtained with the positive control indicate that this did not affect the validity of the test.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was added directly to the dilution water. The test material was weighed into small beakers, then washed into the test bottles using deionised water.
- Controls: Abiotic controls (no inoculum, 1000 mg/L of test material) and blank controls (inoculum, no test material).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: Upon collection, the sludge was washed and centrifuged and the supernatant liquid phase decanted. The solid material was re-suspended in chlorine-free tap water and 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 2 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.42. The activated sludge was used directly after conditioning.
- Initial biomass concentration: 2 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21.6 to 21.8 °C

Dissolved oxygen:

7.39 to 8.91 mg O₂/L (measured during the first 30 seconds after cessation of aeration)

Nominal and measured concentrations:

- Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Infusion bottles and BOD bottles with a special neck
- Material, size, headspace, fill volume: 500 mL infusion bottles, 330 mL BOD bottles.
- Aeration: Compressed air (1 L/min)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5 for the abiotic control, 5 blank controls (one for each of the three test concentrations, one for the abiotic control and one for the positive control) and 1 replicate for each of the three positive control test concentrations
- Biomass loading rate: 2 g/L suspended solids

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
16 mL of synthetic sewage feed (16.0 g peptone, 11.0 g meat extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl₂.2H₂O, 0.2 g MgSO₄.7H₂O, 2.8 g K₂HPO₄ in 1000 mL of deionised water) was added to an adequate volume of the appropriate test material stock solution and filled up with deionised water to 250 mL or 500 mL. At the start of the test, 250 mL of the active sludge inoculum was added to the test vessels (not to the abiotic control).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Respiration inhibition. For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask, after about 3 hours of incubation time the aeration was stopped. The dissolved oxygen concentration was measured for about fifteen minutes with an O₂ electrode, recorded every half minute. The oxygen consumption (in mg O₂/L/minute) was determined from the most linear part of the respiration curve.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 fold

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol 0.4, 4 and 40 mg/L

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:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No confounding effects reported

The average of specific respiration rate of the blank controls was 14.05 mg O₂/L/h/g solid. The coefficient of variation of oxygen uptake rate in untreated replicates was 7.53 %.
The average total respiration rate of the treated parallel mixtures for the test material concentrations at 10, 100 and 1000 mg/L were 14.04, 12.10 and 8.44 mg/L/hour, respectively.
The average respiration rate of the abiotic parallels was 0.46 mg O₂/L/h.
The average inhibition values for 10, 100 and 1000 mg/L were 1.85, 20.58 and 37.23 %, respectively.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 2.69 mg/L 95 % CL 1.74 to 4.15 mg/L

Reported statistics and error estimates:

The oxygen uptake rates (R) were calculated from the measured values, based on the linear part of the graphs of oxygen concentration (values taken in consideration between 2.0 and 7.0 mg/L only) versus time.
R = (Q1 – Q2) / (Δt x 60)
Where:
Q1 = oxygen concentration at the beginning of the selected section of the linear phase (mg/L).
Q2 = oxygen concentration at the end of the selected section of the linear phase (mg/L).
Δt = time interval (minutes)

The percentage inhibition of the total oxygen consumption (IT) was calculated:
IT = 1-((RT – RTA) / RTB) x 100 %
Where:
RT = rate of total oxygen consumption (mg/L/hour).
RTA = rate of total oxygen consumption (mg/L/hour) in the abiotic control
RTB = rate of total oxygen consumption (mg/L/hour) in the blank control

The specific respiration rate (Rs) was calculated as the ratio of the consumed oxygen and the dry weight (g) of sludge per hour (mg/g/h)
Rs = R / SS
Where:
SS = Concentration of suspended solids (g/L)
R = Oxygen uptake rate

The percentage inhibition of oxygen uptake was plotted against logarithm of the test material concentration (inhibition curve). The inhibition curves were plotted for each aeration period of 3 hours.
The 3-hour EC50 and 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software for the positive control. Due to the low inhibition effect of the test material, the EC50 was incalculable statistically; it could only be interpolated from the inhibition graph.

Table 1: Summary of Results

Group	Conc. (mg/L)	Total O₂ consumption rate (mg O₂ /L/h)	Specific respiration rate (mg O₂ /L/h/g solid)	Inhibition (%)	pH†		Test temperature (°C)#	Variation coefficient of O₂ consumption rate (%)
					Start	End
A1	1000	0.96	-	-	7.22	7.30	21.6 / 21.8	-
A2	1000	0.58	-	-	7.22	7.21		-
A3	1000	0.24	-	-	7.22	7.20		-
A4	1000	0.00	-	-	7.24	7.20		-
A5	1000	0.53	-	-	7.22	7.19		-
B1	0.00	13.54	13.54	-	7.75	7.83		7.53
B2	0.00	15.48	15.48	-	7.78	7.86
B3	0.00	13.84	13.84	-	7.91	7.92
B4	0.00	14.65	14.65	-	7.80	7.87
B5	0.00	12.72	12.72	-	7.64	7.88
PC1	0.40	11.43	11.43	29.13	7.79	7.86		-
PC2	4.00	8.58	8.58	47.52	7.80	7.87		-
PC3	40.00	2.46	2.46	87.10	7.80	7.86		-
T1*	10	14.04	14.04	1.85	7.90	8.17		-
T2*	100	12.10	12.10	20.58	7.69	8.21		-
T3*	1000	8.44	8.44	37.23	7.42	7.90		-

A1 to A5 = Abiotic control replicates

B1 to B5 - Blank control replicates

PC1 to PC3 = Positive control replicates

T1 to T3 = Test material concentrations

* Average of five replicates

# Minimum / Maximum temperature measured in the laboratory during the experiment

† Individual pH values of the BOD flasks are reported except for the test material replicates; here the average values are reported

Validity criteria fulfilled:

no

Remarks:

Oxygen uptake rate of the blank controls should be >20 mg O₂/L/h/g; the average for the test was 14.05 mg O₂/L/h/g. This was attributed to an inconsistent ambient temperature of the STP. This was not thought to affect the overall outcome of the results.

Conclusions:

Under the conditions of the test, the EC50 of the test material to activated sludge was determined to be greater than 1000 mg/L.

Executive summary:

The toxicity of the test material to microorganisms was determined in an activated sludge respiration inhibition test conducted in accordance with the standardised guidelines OECD 209 and EU Method C.11. The study was performed under GLP conditions.

Activated sewage sludge (obtained from a domestic sewage treatment plant) was incubated for 3 hours under aeration with the test material at concentrations of 10, 100 and 1000 mg/L. At the end of the incubation period, the oxygen respiration rate was measured over a period of 15 minutes with an oxygen electrode. Blank, abiotic and positive controls were run in parallel for comparison of respiration rates and also to confirm the validity of the test.

The average of specific respiration rate of the blank controls was 14.05 mg O₂/L/h/g solid. The coefficient of variation of oxygen uptake rate in untreated replicates was 7.53 %. The oxygen uptake rate of the blank controls should be >20 mg O₂/L/h/g; however, the high EC50 obtained for the test material and the good results obtained with the positive control indicate that this did not affect the validity of the test.

The average total respiration rate of the treated parallel mixtures for the test material concentrations at 10, 100 and 1000 mg/L were 14.04, 12.10 and 8.44 mg/L/hour, respectively.

The average respiration rate of the abiotic parallels was 0.46 mg O₂/L/h.

The average inhibition values for 10, 100 and 1000 mg/L were 1.85, 20.58 and 37.23 %, respectively.

Under the conditions of the test, the EC50 of the test material to activated sludge was determined to be greater than 1000 mg/L.

Description of key information

3 hour EC50 > 1000 mg/L, activated sludge, OECD 209, EU Method C.11, Oláh (2014)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

In the key study (Oláh 2014), the toxicity of the test material to microorganisms was determined in an activated sludge respiration inhibition test conducted in accordance with the standardised guidelines OECD 209 and EU Method C.11. The study was performed under GLP conditions.

The study was therefore assigned a reliability score of 1 in accordance with the principles for assessing data quality as defined in Klimisch et al. (1997).

Activated sewage sludge (obtained from a domestic sewage treatment plant) was incubated for 3 hours under aeration with the test material at concentrations of 10, 100 and 1000 mg/L. At the end of the incubation period, the oxygen respiration rate was measured over a period of 15 minutes with an oxygen electrode. Blank, abiotic and positive controls were run in parallel for comparison of respiration rates and also to confirm the validity of the test.

The average of specific respiration rate of the blank controls was 14.05 mg O₂/L/h/g solid. The coefficient of variation of oxygen uptake rate in untreated replicates was 7.53 %. The oxygen uptake rate of the blank controls should be >20 mg O₂/L/h/g; however, the high EC50 obtained for the test material and the good results obtained with the positive control indicate that this did not affect the validity of the test.

The average total respiration rate of the treated parallel mixtures for the test material concentrations at 10, 100 and 1000 mg/L were 14.04, 12.10 and 8.44 mg/L/hour, respectively.

The average respiration rate of the abiotic parallels was 0.46 mg O₂/L/h.

The average inhibition values for 10, 100 and 1000 mg/L were 1.85, 20.58 and 37.23 %, respectively.

Under the conditions of the test, the EC50 of the test material to activated sludge was determined to be greater than 1000 mg/L.