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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-14 to 2016-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A saturated solution was prepared: 180 mg/L were weighed out in ameasuring flask, filled up with demineralised water and stirred overnight. The saturated solution was filtered (45 µm). The concentration was determined by measurement of DOC (dissolved organic carbon) and was 43.7 mg/L. The DOC concentration was used to calculate the required volumes of saturated solution in the test vessels of the test item concentrations 0.1 mg/L (0.725 mL) and 0.56 mgIL (4.05 mL). The concentrations 3.2 mg/L, 18 mgIL and 100 mg/L was weighed out onto object slides and transferred with these into the Erlenmeyer flasks. 200 mL demineralised water was added and stirred overnight.
- Controls:
Test medium without test or reference item
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected:
Municipal sewage treatment plant of 31137 Hildesheim, Germany
- Preparation of inoculum for exposure:
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.
- Initial biomass concentration:
Dry sludge concentration: 3.05 g/L, corresponding to 1.53 g/L in the test vessel
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
n.a.
Test temperature:
18 - 22°C
pH:
7.5 ± 0.5
Dissolved oxygen:
> 60-70%
Salinity:
n.a.
Conductivity:
n.a.
Nominal and measured concentrations:
0.1, 0.56, 3.2, 18 and 100 mg/L.
The concentration was determined by measurement of DOC (dissolved organic carbon). The DOC concentration was used to calculate the required volumes of saturated solution in the test vessels of the test item concentrations 0.1 mg/L and 0.56 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: size: 1000 mL, fill volume: 500 mL
- Aeration: by shaking
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 3.05 g/L, corresponding to 1.53 g/L in the test vessel

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralised water

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration Inhibition measured after 3h exposure
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5.6
- Range finding study
- Test concentrations: 1st preliminary test: 10-100-1000 mg/L, 2nd preliminary test: 10 mg/L
- Results used to determine the conditions for the definitive study: Based on these results the definitive study was carried out with 5 concentrations within the range 0.1 - 100 mg/L in a geometrical series and with a dilution factor of 5.6 (above 100 mg/L, no increase in inhibition was determined in the 1st preliminary test).
Reference substance (positive control):
yes
Remarks:
Copper (ll) sulphate pentahydrate p.a.
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
1.93 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
14.9 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
0.56 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 107 mg/L; 95% CI = 103-111 mg/L; recommended range of validity: 53-155 mg/L
Reported statistics and error estimates:
One Way Analysis of Variance (ANOVA) and DUNNETT'S test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism.

Validity of the study:

The study is valid as:

- the mean specific oxygen uptake rate of the control replicates was 21. 7 mg 02/g·h (validity

criterion: should be > 20 mg 02/g·h)

- the coefficient of variation of the oxygen uptake rates in the control replicates was 4.2 %

(validity criterion: < 30 %)

- the EC50 of the reference item was 107 mg/L and in the range of 53 - 155 mg/L

Table 1: Measured Oxygen uptake rate data

 Treatment (mg a.i./L) [measured conc. used]

Exposure time [3 h]

Oxygen uptake rate [mg O2/L ×h]

% Inhibition

Control (dilution water only)

Replicate #1

33.6

-

Replicate #2

34.4

-

Replicate #3

33.2

-

Replicate #4

31.6

-

Replicate #5

31.2

-

Replicate #6

31.2

-

0.1

Replicate #1

33.2

-2

Replicate #2

33.2

-2

Replicate #3

33.2

-2

Replicate #4

33.6

-3

Replicate #5

32.4

0

0.56

Replicate #1

32.4

0

Replicate #2

32.4

0

Replicate #3

30.8

5

Replicate #4

29.6

9

Replicate #5

31.2

4

3.2

Replicate #1

25.6

21

Replicate #2

26.0

20

Replicate #3

27.6

15

Replicate #4

28.0

14

Replicate #5

28.0

14

18

Replicate #1

14.8

54

Replicate #2

15.2

53

Replicate #3

14.8

54

Replicate #4

15.2

53

Replicate #5

14.8

54

100

Replicate #1

10.8

67

Replicate #2

10.4

68

Replicate #3

10.8

67

Replicate #4

10.4

68

Replicate #5

11.2

66

Validity criteria fulfilled:
yes
Conclusions:
The present study was conducted according to OECD Test Guideline 209 in order to determine the respiration inhibition mediated by 2,4-Bismaleimidotoluene. Non-adapted activated sludge from a municipal sewage plant was incubated with 2,4-Bismaleimidotoluene for 3 h with the following concentrations: 0.1, 0.56, 3.2, 18 and 100 mg/L. The control was the test medium without test or reference item. Copper (ll) sulphate pentahydrate was used as reference substance and was incubated with 58, 100 and 180 mg/L. 2,4-Bismaleinimidotoluene is considered to be not toxic up to a concentration of 0.56 mg/L.
The following EC values and NOEC were determined:
EC10 = 1.93 mg/L; 95 % CI = 1.62-2.30
EC50 = 14.9 mg/L; 95 % CI = 13.1-17.1
NOEC = 0.56 mg/L: 95 % CI = -----

Description of key information

- Toxicity to microorganisms study; according to OECD 209; RL 1; GLP; EC50 = 14.9 mg/L, EC10 = 1.93 mg/L and NOEC = 0.56 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
14.9 mg/L
EC10 or NOEC for microorganisms:
1.93 mg/L

Additional information

The toxicity of 2,4-Bismaleimidotoluene to microorganisms was investigated in an activated sludge respiration inhibition test according to OECD guideline 209 (2010), respiration inhibition test using an inoculum obtained from non-adapted, activated sludge from a predominantly domestic sewage treatment plant over 48 h. The test was carried out under static conditions with the test item concentrations 0.1 -0.56 - 3.2 - 18 - 100 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. In order to check the activity of the test system and the test conditions a reference test was carried out with copper (ll) sulphate pentahydrate as reference item and the reference toxicity was determined.

The EC 50-value for the reference item was 107 mg/L. The mean inhibition of respiration for the test item replicates ranged from -2 % to 67 %. The EC10 was determined to be 1.93 mg/L, the EC50 was 14.9 mg/L and the NOEC was determined to be 0.56 mg/L.

Since, the coefficient of variation of the respiration rates of the control was < 30% and the oxygen uptakes rates of the control replicates were > 20 mg O2/g*h and the EC50-value of the reference was in the range of 53-155 mg/L the validity criteria were fulfilled. The test item had no inhibitory effect on oxygen uptake of sludge microorganisms up to a concentration of 0.56 mg/L, thus, the NOEC of 2,4-Bismaleimidotoluene is 0.56 mg/L. The determined EC50 is 14.9 mg/L (95 % CI = 13.1 -17.1) and the determined EC10 is 1.93 mg/L (95 % CI = 1.62 -2.30).