trans-1,4-bis(isocyanatomethyl)cyclohexane

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:

23 February 2016 - 25 March 2016

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 (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

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

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-Litre test bottles were filled with 200 mL 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	FORTIMO^TM1,4-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).

Description of key information

In a Respiration Inhibition test (OECD 209) the substance was not toxic to STP bacteria at or below a loading rate of 3.2 mg/L. The ELR50 was at a loading rate of 17 mg/L (95% confidence interval: 14 – 21 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

17 mg/L

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).