Octahydro-2H-1-benzopyran-2-one

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:

31 August 2016 - 25 November 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:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008 (ammended by EC no. 2016/266 of 7 Dec 2015)

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:

Water solubility: 39953 mg/L at 24°C

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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 (tap water purified by reverse osmosis) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL 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.
- Controls: Test medium without test item and treated in the same way as the test item solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Inoculum: A sample of activated sludge was obtained the day before the start of the tests from a 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 (3.0 g/L of sludge, as used for the test). 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. The pH was 7.9 on the day of testing.
- Initial biomass concentration: 3 g suspended solids/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19- 22 °C

pH:

7.0-7.3 (start), 7.4- 8.2 (end)

Nominal and measured concentrations:

Nominal concentrations (final test): 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM (Definitive test):
- Test vessel: 1L all-glass, open bottles, fill volume: 500 mL
- Aeration: yes; clean, oil-free compressed air - aeration was 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
- Suspended solids concentration in final test solution: 1.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis (Millipore Corp., USA)
- Test medium: adjusted ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature - continuously in a control vessel, pH - at the start and the end of the test period

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: rate of oxygen consumption for approx. 10 minutes after 3 hours of exposure to the test substance.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
* Range finding study
- Test concentrations (loading rates): 10, 100 and 1000 mg/L
- Method: The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, a blank control (6 replicates) and a nitrification control (2 replicates) were included. Furthermore, an abiotic control (1 replicate) and the highest loading rate with a nitrification inhibitor (3 replicates) were tested.
- Results used to determine the conditions for the definitive study: Yes, the combined limit/range-finding test showed 7, 16 and 65% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was between a loading rate of 100 and 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (nominal concentrations of 1.0, 3.2, 10 and 32 mg/L)

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate)

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate)

Basis for effect:

inhibition of total respiration

Details on results:

- No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 320 mg test substance per litre. At 1000 mg/L the inhibitory effect of the test substance on aerobic waste water (activated sludge) bacteria was 35% inhibition.
- In the combined limit/range-finding test there was no significant oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. Based on the results of the total and heterotrophic respiration, nitrification inhibition was calculated to be 93%. There were no spurious results (i.e. bi-phasic concentration related respiration curves, distorted dose-responses or unexpectedly low EC50 values) observed in the combined limit/range-finding. Therefore, N-allylthiourea was not used in the final test.
- The effect in the final test was lower than in the combined limit/range-finding test. It should be noted that the pH in the combined limit/range-finding test was lower than in the final test, which might indicate a difference in buffer capacity between the two systems. Furthermore, the effect in the combined limit/range-finding test may be attributed to an acid effect, due to hydrolysis of the test substance. In general the buffering capacity of the sewage treatment plant is sufficient to account for such effects and therefore, the results of the final test are used to derive the NOEC and EC50 for the test substance.
- Experimental conditions throughout the test complied with the requirements laid down in the study plan.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 6.05 mg/L
- Other: these results indicate that the batch of activated sludge showed normal sensitivity.

Reported statistics and error estimates:

Determination of NOEC: 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 (Williams Multiple Sequential t-test Procedure, 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		7.1 – 7.3	8.1 – 8.2	42.02	28.01
T1	10	7.0 – 7.1	8.0 – 8.2	44.50	29.67	-5.91
T2	32	7.0 – 7.1	8.1 – 8.2	45.45	30.30	-8.17
T3	100	7.0	8.1 – 8.2	43.05	28.70	-2.46
T4	320	7.0	7.9 – 8.0	44.94	29.96	-6.95
T5	1000	7.0	7.4 – 7.6	27.41	18.27	34.78 *

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

* Statistically significantly different compared to control

Validity criteria fulfilled:

yes

Remarks:

For details see 'Overall Remarks' section

Conclusions:

Under the conditions of the test the test substance was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 320 mg/L (NOEC). In the final test, the highest tested concentration (loading rate of 1000 mg/L) caused 35% inhibition of the total respiration rate. The EC50 of the test substance was therefore determined to be > 1000 mg/L.

Executive summary:

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209 and under GLP, microorganisms in activated sludge were exposed to a blank control and 5 concentrations of the test substance at loading rates of 10, 32, 100, 320 and 1000 mg test substance/L. The EC50 of the test substance was determined to be > 1000 mg/L (based on loading rate). No statistically significant inhibition of respiration was observed at or below 320 mg/L (NOEC). The study met the acceptability criteria prescribed by the study plan and was considered valid.

Description of key information

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209 and under GLP, microorganisms in activated sludge were exposed to a blank control and 5 concentrations of the test substance at loading rates of 10, 32, 100, 320 and 1000 mg test substance/L. The EC50 of the test substance was determined to be > 1000 mg/L (based on loading rate). No statistically significant inhibition of respiration was observed at or below 320 mg/L (NOEC). The study met the acceptability criteria prescribed by the study plan and was considered valid.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

320 mg/L