1,3-Cyclohexanedimethanamine, N1,N3-bis(2-methylpropylidene)-

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:

2011-04-20 to 2011-04-20

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Based on the results of a performed preliminary informatory range-finding test the test item was investigated in five concentration levels 62.5, 125, 250, 500 and 1000 mg/L. Because of the handling difficulties (preliminary information from the stability and solubility properties), the adequate amounts of the test item were weighted in a small inert plastic dish (“plate”: to allow the contact between the inoculum and the whole test item amount) and this plastic dish containing the adequate test item amount was thrown into each test flask (nearly simultaneously (< 30 sec.)) with the inoculum addition (just a short pH measurement was performed before the inoculation) before the inoculation and the subsequent calculations referred to the initial weighed nominal concentration.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Preparation of inoculum for exposure: and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. 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 amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre (on dry weight basis). The activated sludge was not used on the day of the collection but continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18.0 - 21.4 °C

pH:

7.8 - 9.23

Nominal and measured concentrations:

62.5, 125, 250, 500 and 1000 mg/L (nominal)
No measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer bottles of approximately 300 mL volume
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): five concentrations
- No. of vessels per control (replicates): six replicates
- No. of vessels per reference control (replicates): three concentrations with three replicates
- No. of vessels per nitrification control (replicates): three replicates

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: the test was performed without pH adjustment

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study:
Pre-experiments were performed to determine the solubility of the test item in the used aqueous system, to investigate the test item effect on the pH within the test system and to select suitable methods for the preparation of a stock solution and for the dosage of the test item in the test medium.
The preliminary range-finding test one test container from the concentration of 10 mg/L, one container from the concentration of 100 mg/L, triplicates at the highest test item concentration (1000 mg/L), blank, abiotic (three abiotic controls with the highest concentration of the test substance: 1000 mg/L) and reference controls were included. The nitrification potential of sludge was examined with additional control mixture that contained N-allylthiourea at 11.6 mg/L. In the preliminary range-finding test the test item showed a pronounced inhibitory effect at the concentration of 1000 mg/L. Therefore investigation of a concentration series of the test item was considered as necessary. The abiotic control group did not show significant oxygen uptake (some oxygen uptake was detected in the range of the biological variability of the test system), therefore in the main test the abiotic control was not repeated. In the preliminary test the nitrification respiration was insignificant and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification is occurring. Therefore the main test was performed in one set of test vessels, without ATU addition.
In the main test (for informative reason) a nitrification control group (containing N-allylthiourea) was investigated with three parallels.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

94.96 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95 % CI: 0.001–218.82 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

375.38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95 % CI: 115.97 –7592.08 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

925.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95 % CI: 408.38 – 48638225.74 mg/L

Details on results:

Inhibition of Respiration Rate:
An additional nitrification control was examined in the main test with three parallels to check the possible nitrification potential of the applied activated sludge batch. With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 52.60 mg/Lh, the heterotrophic respiration (RH) was 50.48 mg/Lh, the nitrification respiration (RN): 2.12 mg/Lh was calculated according to the following equation: RN= RT-RH. The obtained 2.12 mg/Lh was insignificant [lower than the 5 % of RT (2.63 mg/Lh) in blank controls]. According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 6.03 mg/L, (95 % confidence limits: 4.88 - 7.41).
- Other: The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. Each concentration of the 3,5-Dichlorophenol reference item was tested in three parallels. In comparison to the blank controls the specific respiration rate of the activated sludge was inhibited by 19.31 % at the lowest concentration of 2 mg/L, at the nominal concentrations of 7 and 24.5 mg/L, the respiration rate was inhibited by 54.90 % and 86.17 %, respectively.

Reported statistics and error estimates:

The ECx values of the test item and the reference item and their confidence limits were calculated using Probit analysis. The analysis was done using the SPSS statistical software program. For the determination of the LOEC and NOEC the calculated specific respiration rates were tested on significant differences to the control values by Bonferroni t-Test by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC50 value of Incorez 397 test item was determined as 375.38 mg/L (95 % confidence limits: 115.97 – 7592.08 mg/L), and the EC10 as 94.96 mg/L.

Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item Incorez 397 on the activity of activated sludge by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Nominal concentrations of 2.5, 125, 250, 500 and 1000 mg Incorez 397/L were investigated. At the concentrations of 62.5, 125 and 250 mg/L the observed-calculated specific respiration rates were slightly lower than the specific respiration rates of the blank controls, however, were in the same range (within the biological variability of the applied system) and were considered biologically as not significant. The inhibition of the specific respiration rates compared to the respiration rates of the blank controls was 12.02 % at 62.5 mg/L, 15.19 % at 125 mg/L and 13.65 % at 250 mg/L. The test item showed significant inhibitory effect at the concentrations of 500 mg/L (64.77 %) and at 1000 mg/L (88.98 %). The EC50 was determined to be 375.38 mg/L and the EC10 to be 94.96 mg/L, repectively.

Description of key information

The toxicity to microorganisms of Incorez 397 was tested according to OECD guideline 209 and EU method C.11. Activated sludge was exposed at nominal concentrations of 62.5, 125, 250, 500 and 1000 mg /L. The EC50 was determined to be 375.38 mg/L and the EC10 was 94.96 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

375.38 mg/L

EC10 or NOEC for microorganisms:

94.96 mg/L

Additional information

The purpose of the 3-hour test was to evaluate the influence of the test item Incorez 397 on the activity of activated sludge by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Nominal concentrations of 2.5, 125, 250, 500 and 1000 mg Incorez 397/L were investigated. At the concentrations of 62.5, 125 and 250 mg/L the observed-calculated specific respiration rates were slightly lower than the specific respiration rates of the blank controls, however were in the same range (within the biological variability of the applied system) and were considered biologically as not significant. The inhibition of the specific respiration rates compared to the respiration rates of the blank controls was 12.02 % at 62.5 mg/L, 15.19 % at 125 mg/L and 13.65 % at 250 mg/L. The test item showed significant inhibitory effect at the concentrations of 500 mg/L (64.77 %) and at 1000 mg/L (88.98 %). The EC50 was determined to be 375.38 mg/L and the EC10 was 94.96 mg/L.