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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-04-2016 till 26-04-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, all validity criteria fulfilled, GLP
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
-
Vehicle:
no
Details on test solutions:
A preliminary test (non GLP) was carried out with the following test item concentrations of TETRAMEEN 2HT: 10 - 100 - 1000 mg/L with two replicates each and additionally two replicates with 1000 mg/L to study the inhibition of the nitrification using Allylthiourea ATU (Final concentration: 11.6 mg/L). The pH was adjusted in one replicate with the concentration of 1000 mg/L.

Based on the results of the preliminary test it was concluded that the heterotrophic respiration as well as the nitrification were not inhibited. Therefore the definitive study was carried out with the concentration of 1000 mg/L in 5 replicates (limit test to determine the NOEC). The test item was weighed out directly in the Erlenmeyer flasks and was stirred overnight.
Triplicates of the control without test item were included at the beginning and at the end of the test. (6 controls in total)
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test System Activated sludge
Origin Municipal sewage treatment plant of 31137 Hildesheim, Germany
Reason for the selection Non-adapted activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.

Pretreatment The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.

Date of receipt 2016-04-25

Dry sludge concentration 2.76 g/L (corresponding to 1.38 g/L suspended solids in the test vessels)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20.6 °C
pH:
7.36
Dissolved oxygen:
The mean specific oxygen uptake rate of the control replicates was 26.6 mg O2/g.h (validity criterion: should be ≥ 20 mg O2/g.h).
Salinity:
Fresh water
Nominal and measured concentrations:
The test item was weighed out directly in the Erlenmeyer flasks and was stirred overnight.
Details on test conditions:
Test Method Static
Test duration 3 h
Test vessels 1000 mL Erlenmeyer flasks, DIN 12380
Nutrient solution Synthetic waste water according to OECD-Guideline 209
Volume of the 500 mL
test medium
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
UVCB, 100% a.i.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
From the oxygen depletion the oxygen uptake rates were calculated in mg O2/Lh, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O2/gh. The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison to the oxygen uptake rates of the control.
Results with reference substance (positive control):
In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 104 mg/L. The validity criteria of the test guideline were fulfilled.
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α=0.05.

The EC-value of the reference item was calculated by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

Software Excel, MICROSOFT CORPORATION
GraphPad Prism, GRAPHPAD SOFTWARE, INC.
SigmaPlot (windows), SPSS INCORPORATION

All data were computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

Table7:      Oxygen Uptake Rates, Inhibition of the Respiration of Control, Test and
Reference Item
Concentrations

Test and Reference Item Concentration

[mg/L]

Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]

Inhibition


[%]

Mean Inhibition

[%]

Control

1

31.6

-

-

2

37.2

3

36.8

4

34.8

5

38.4

6

41.6

Test item
1000

1

38.8

-6

-5

2

35.6

3

3

40.4

-10

4

38.4

-5

5

40.0

-9

Reference item
58

1

27.6

25

25

2

27.6

25

3

28.0

24

100

1

18.8

49

48

2

19.2

48

3

19.2

48

180

1

9.6

74

74

2

9.6

74

3

9.6

74

Repl. = Replicate        

Coefficient of variation of oxygen uptake rates of the control: 9.2%

Suspended solids in the test vessel: 1.38 g/L

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item TETRAMEEN 2HT is ≥ 1000 mg/L.

TETRAMEEN 2HT caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item TETRAMEEN 2HT (batch number: 890000394200) from 2016-04-25 to 2016-04-26, with the definitive exposure phase on 2016-04-26 at the test facility. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit test item concentration 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates was -5 %. No nitrification inhibition was observed at 1000 mg/L in the preliminary test and this endpoint was therefore not evaluated in the definitive test.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 104 mg/L. The validity criteria of the test guideline were fulfilled.

Description of key information

The NOEC of the test substance Tetrameen 2HT is  ≥ 1000 mg/L.

Tetrameen 2HT caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test substance concentration of 1000 mg/L.  No nitrification inhibition was observed at 1000 mg/L in the preliminary test and this endpoint was therefore not evaluated in the definitive test.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The NOEC of the test item Tetrameen 2HT is ≥1000 mg/L.

 

Tetrameen 2HTcaused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test substance concentration of 1000 mg/L. The test substance was weighed out directly in Erlenmeyer flasks used for the test and was stirred overnight. The validity criteria were fulfilled:

·      The mean specific oxygen uptake rate of the control replicates was 26.6 mg O2/g×h (validity criterion:should be³ 20 mg O2/g×h).

·      The coefficient of variation of the oxygen uptake rates in the control replicates was 9.2 % (validity criterion: < 30 %).

·      The EC50 of the reference substance was 104 mg/L and therefore in the range of 53 - 155 mg/L.