Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 2016 - 25 March 2016
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:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No. 440/2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C 11:"Biodegradation: Activated sludge respiration inhibition test"
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
2007
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Stability at higher temperatures: yes, maximum temperature: 40°C
- Highly reactive to water: yes
- Highly reactive to oxygen: not indicated
- Volatile: not indicated
- Solubility in water: not available
- Stability in water: no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-Litre1 test bottles were filled with 200 mL¹ of test item mixtures in Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. Within 35 minutes synthetic medium, Milli-RO water and sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge
Details on inoculum:
- Source: municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: the sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids. The pH was 7.6 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g/L
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.8 - 20.8°C
pH:
7.6-8.1
Nominal and measured concentrations:
Nominal test concentrations: 1.0, 3.2, 10, 32, 100 and 320 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessels: all glass open bottles/vessels
- Air supply: clean, oil-free air
- Aeration: continuously and adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Nitrification inhibitor used: no

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

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Illumination: not specified

EFFECT PARAMETERS MEASURED: oxygen consumption at t=3 h. for 10 minutes; pH at t=0 and t=3 h.; temperature of medium continuously in a temperature control vessel(s).
- Method of oxygen recording: determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GOSystemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (to check activated sludge for sensitivity)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
3.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
EC50: 3.8 mg/L, 95% CI: 1.9 - 7.6 mg/L
- Other: calculation of the EC50 value was based on non-linear regression analyses with the total respiration rate versus the corresponding concentrations of the item.
Reported statistics and error estimates:
Calculation of ELRx values were based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding loading rates of the test item. For NOELR determination, an effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller).

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1            Final test – Overview of the results

Treatment

Loading rate

(mg/L)

pH

Mean respiration rate

% Inhibition of the respiration

rate

(mean value)

Start

End

(mg O2/L h)

(mg O2/g h)¹

Blank control

 

7.4 - 7.5

7.6 - 7.8

39.65

26.43

 

T1

1.0

7.4

7.7

41.74

27.83

-5.29

T2

3.2

7.4 - 7.5

7.7 - 7.8

37.64

25.10

5.06

T3

10

7.4

7.7 - 7.8

25.39

16.93

35.96*

T4

32

7.4

7.9 - 8.0

12.22

8.14

69.19*

T5

100

7.4

7.9 - 8.0

4.59

3.06

88.43*

T6

320

7.3 - 7.4

8.1

0.57

0.38

98.56*

¹)The amount of suspended solids in the final test mixture was 1.5 g/L.

*Statistically significantly different compared to control

Table2            Effect parameters

Parameter

FORTIMOTM1,4-H6XDI

Loading rate (mg/L)

NOELR

3.2

ELR10

3.4 (2.0 - 4.8 mg/L)

ELR20

5.9 (4.0 – 7.8 mg/L)

ELR50

17 (14 - 21 mg/L)

(  ) Between brackets the 95% confidence intervals are given.

Validity criteria fulfilled:
yes
Remarks:
1) Mean blank control oxygen uptake rate > 20 mg oxygen/gram of activated sludge in an hour. 2) The EC50 of the reference substance was in the accepted range of 2-25 mg/L for total respiration.
Conclusions:
Under the conditions of this present test FORTIMOTM 1,4-H6XDI was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 3.2 mg/L (NOELR).
- The ELR10 was at a loading rate of 3.4 mg/L (95% confidence interval: 2.0 – 4.8 mg/L).
- The ELR20 was at a loading rate of 5.9 mg/L (95% confidence interval: 4.0 – 7.8 mg/L).
- The ELR50 was at a loading rate of 17 mg/L (95% confidence interval: 14 – 21 mg/L).
Executive summary:

The influence of 1,4 -H6XDI on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

 Because of insufficient solubility of the test substance, 1-Liter test bottles were filled with 200 mL of test item mixtures with initial loading rates of 2.5 times the final loading rate. Optimal contact between the test item, test organisms and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes. The final test was performed based on the result of a preceding combined limit/range-finding test. Loading rates, ranging from 1.0 to 320 mg/L, increasing with a factor 3.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 3.2 mg test substance per liter. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 36% inhibition at 10 mg/L to 99% at 320 mg/L. The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the study plan and was considered valid. An ELR50 of 17 mg/L was found. In conclusion, the test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 3.2 mg/L (NOELR).

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose:
read-across source
Analytical monitoring:
no
Vehicle:
no
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
3.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
EC50: 3.8 mg/L, 95% CI: 1.9 - 7.6 mg/L
- Other: calculation of the EC50 value was based on non-linear regression analyses with the total respiration rate versus the corresponding concentrations of the item.
Reported statistics and error estimates:
Calculation of ELRx values were based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding loading rates of the test item. For NOELR determination, an effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller).

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1            Final test – Overview of the results

Treatment

Loading rate

(mg/L)

pH

Mean respiration rate

% Inhibition of the respiration

rate

(mean value)

Start

End

(mg O2/L h)

(mg O2/g h)¹

Blank control

 

7.4 - 7.5

7.6 - 7.8

39.65

26.43

 

T1

1.0

7.4

7.7

41.74

27.83

-5.29

T2

3.2

7.4 - 7.5

7.7 - 7.8

37.64

25.10

5.06

T3

10

7.4

7.7 - 7.8

25.39

16.93

35.96*

T4

32

7.4

7.9 - 8.0

12.22

8.14

69.19*

T5

100

7.4

7.9 - 8.0

4.59

3.06

88.43*

T6

320

7.3 - 7.4

8.1

0.57

0.38

98.56*

¹)The amount of suspended solids in the final test mixture was 1.5 g/L.

*Statistically significantly different compared to control

Table2            Effect parameters

Parameter

FORTIMOTM1,4-H6XDI

Loading rate (mg/L)

NOELR

3.2

ELR10

3.4 (2.0 - 4.8 mg/L)

ELR20

5.9 (4.0 – 7.8 mg/L)

ELR50

17 (14 - 21 mg/L)

(  ) Between brackets the 95% confidence intervals are given.

Validity criteria fulfilled:
yes
Remarks:
1) Mean blank control oxygen uptake rate > 20 mg oxygen/gram of activated sludge in an hour. 2) The EC50 of the reference substance was in the accepted range of 2-25 mg/L for total respiration.
Conclusions:
Under the conditions of this present test FORTIMOTM 1,4-H6XDI was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 3.2 mg/L (NOELR).
- The ELR10 was at a loading rate of 3.4 mg/L (95% confidence interval: 2.0 – 4.8 mg/L).
- The ELR20 was at a loading rate of 5.9 mg/L (95% confidence interval: 4.0 – 7.8 mg/L).
- The ELR50 was at a loading rate of 17 mg/L (95% confidence interval: 14 – 21 mg/L).
This result is read across to 1,3-H6XDI.
Executive summary:

The influence of 1,4 -H6XDI on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

 Because of insufficient solubility of the test substance, 1-Liter test bottles were filled with 200 mL of test item mixtures with initial loading rates of 2.5 times the final loading rate. Optimal contact between the test item, test organisms and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes. The final test was performed based on the result of a preceding combined limit/range-finding test. Loading rates, ranging from 1.0 to 320 mg/L, increasing with a factor 3.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 3.2 mg test substance per liter. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 36% inhibition at 10 mg/L to 99% at 320 mg/L. The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the study plan and was considered valid. An ELR50 of 17 mg/L was found. In conclusion, the test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 3.2 mg/L (NOELR). This result is read across to 1,3-H6XDI.

Description of key information

The substance 1,4 -H6XDI was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 3.2 mg/L. This result is read across to 1,3-H6XDI.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
3.2 mg/L

Additional information

The influence of 1,4 -H6XDI on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

 Because of insufficient solubility of the test substance, 1-Liter test bottles were filled with 200 mL of test item mixtures with initial loading rates of 2.5 times the final loading rate. Optimal contact between the test item, test organisms and test medium was ensured applying continuous aeration and stirring during the3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.The final test was performed based on the result of a preceding combined limit/range-finding test. Loading rates, ranging from 1.0 to 320 mg/L, increasing with a factor 3.2 were tested. Five replicates per loading rate and sixreplicates for an untreated control group were tested.No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 3.2 mg test substance per liter. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 36% inhibition at 10 mg/L to 99% at 320 mg/L.The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.The study met the acceptability criteria prescribed by the study plan and was considered valid.An ELR50 of 17 mg/L was found. In conclusion, the test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 3.2 mg/L (NOELR).This result is read across to 1,3-H6XDI.