[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-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:

2019-10-23 to 2020-01-14

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Activated sludge collected from the aeration tank of BRIANZACQUE wastewater treatment plant treating domestic wastewater.
10 litres of activated sludge were collected and transferred to Test Facility where they were immediately set under aeration and shaking in dark conditions. The temperature of the sludge on receipt was 15.0 °C.
The sludge was washed by centrifugation, the supernatant liquid phase decanted and the solid material re-suspended in chlorine-free tap water. This procedure was repeated two times. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated as 6.63%.
Based on this ratio, calculated amounts of wet sludge were suspended in the test water (free-chlorine tap water) to obtain a concentration equivalent to 3.0 g dry weight per litre.
The obtained suspension was held under constant aeration, at room temperature and dark conditions for one day prior to the test initiation.
50 mL per litre of a synthetic feeding prepared immediately before use were added to the sludge inoculum prepared as described above.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

N/A

Hardness:

not reported

Test temperature:

20.2 – 21.7°C

pH:

Without ATU:
7.35 – 7.43 at the test start in test item bottles.
7.23 – 7.64 at the test end.

with ATU:
6.86 – 7.36 at the test start in test item bottles.
7.32 – 7.60 at the test end.

Dissolved oxygen:

7.0 - 2.0 mg O2/L (When the oxygen concentration dropped below 2.0 mg O2/L, the measurement was stopped in accordance with the reference guidelines. )

Salinity:

N/A

Conductivity:

not reported

Nominal and measured concentrations:

Nominal: 42.7, 93.9, 206.6, 454.5 and 1000.0 mg/L.

Details on test conditions:

Since a preliminary test have shown a 60.3% of respiration inhibition at the highest concentration (1000 mg/L of test item), a definitive test at five concentrations was performed. In addition, during the preliminary test, two additional blank bottles with ATU were performed (N-allylthiourea), run in parallel to the blank bottles without ATU.
The difference between the respiration rate of the two series of blank bottles (without and with ATU), showed that the respiration rate due to the nitrification respiration is significant (> 5% of RT), therefore two series of test bottles in definitive test were prepared, one series without ATU and one series with ATU.
This method permitted to measure the oxygen uptake due to the total respiration (RT) and the oxygen uptake due to the heterotrophic respiration (RH). The relationship between total respiration and heterotrophic respiration allows to calculate the oxygen uptake due to nitrification respiration (RN).

Definitive test without ATU (total respiration RT):
Test bottles including negative controls, reference item, test item and abiotic controls were prepared as described below.
The oxygen consumption measurements for each bottle were performed after 3 hours of incubation.
Test item and sludge inoculum (except for abiotic controls) were poured into the test bottles immediately prior to the incubation.
Negative control: Two negative controls (synthetic feeding, demineralised water and sludge inoculum without test item) were prepared and used.
Reference item: Reference item 3,5-dichlorophenol was tested at three concentrations, 1 mg/L, 5 mg/L and 25 mg/L, under the same test conditions, in order to check the sensitivity of sludge inoculum. A stock solution at 1 g/L was prepared before the start of the test (0.1000 g into 100 mL of demineralised water), the pH was 7.02. The test concentrations were prepared by taking adequate aliquots of stock solution and diluting them in reference item bottles (see following Table).
Test item: Five nominal concentrations of test item, 42.7, 93.9, 206.6, 454.5 and 1000.0 mg/L, were tested. Since test item was not soluble in water, the test item concentrations bottles were prepared adding adequate weight of test item into each test bottle. Results are related to the nominal concentration of test item.
Abiotic control: To measure the abiotic oxygen consumption, one abiotic control triplicates at the highest tested concentration of test item (1000 mg/L) were tested

Definitive test with ATU (heterotrophic respiration RH):
Test bottles including negative controls, reference item and test item were prepared as described below.
The oxygen consumption measurements for each bottle were performed after 3 hours of incubation.
Test item and sludge inoculum were poured into the test bottles immediately prior to the incubation.
In addition in each test bottles were poured 2.5 mL of a stock solution concentration of N-allylthiourea at 2.32 g/L (0.2320 g in 100 mL) to obtained a final concentration equal to 11.6 mg ATU/L.
This ATU concentration is known to be sufficient to cause 100% inhibition of nitrification in a nitrifying activated sludge.
Negative control: Two negative controls (synthetic feeding, demineralised water and sludge inoculum without test item) were prepared and used.
Reference item: Reference item 3,5-dichlorophenol was tested at three concentrations, 1.0, 5.0 mg/L and 25.0 mg/L, under the same test conditions, in order to check the sensitivity of sludge inoculum. A stock solution at 1 g/L was prepared before the start of the test (0.1 g into 100 mL of demineralised water), the pH was 7.03. The test concentrations were prepared by taking adequate aliquots of stock solution and diluting them in reference item bottles.
Test item: Five nominal concentrations of test item, 42.7, 93.9, 206.6, 454.5 and 1000.0 mg/L, were tested. Since test item was not soluble in water, the test item concentrations bottles were prepared adding adequate weight of test item into each test bottle. Results are related to the nominal concentration of test item.

Experimental conditions:
Tightly closed plastic bottles (1500 mL capacity) were used and sludge oxygen demand was satisfied stirring the bottles with an orbital shaker at 200 rpm, as suggested by the OECD guideline No. 209 (2010) when the test item generates slightly foam.
During the incubation period, test bottles were maintained in dark conditions and test temperature was in the range 20.2 – 21.7°C with a mean value of 21.1°C and a standard deviation of 0.5°C. The recorded values were corrected according to the data logger correction factor.
During the incubation period the dissolved oxygen was greater than 60% of saturation (95%).
The pH was determined in the test item and reference item concentration bottles, in the abiotic controls and in the negative controls at the end of the test (after 3 hours of incubation), while was determined only in the test item bottles at the start of the test (before inoculum).

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

206.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

454.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

903.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

Definitive test without ATU (total respiration RT):
- Three abiotic controls were tested to measure the abiotic oxygen consumption. After 3 hours of exposure, abiotic controls did not show any significant oxygen consumption.
- The oxygen consumption rate (mg O2/L/h) was determined from the linear part of the respiration curve, approximately in the range of 7.0 - 2.0 mg O2/L.
When the oxygen concentration dropped below 2.0 mg O2/L, the measurement was stopped in accordance with the OECD guideline.
3 h EC50 > 1000 mg/L
NOEC = 454.5 mg/L


Definitive test with ATU (heterotrophic respiration RH):
- The oxygen consumption rate (mg O2/L/h) was determined from the linear part of the respiration curve, approximately in the range of 7.0 - 2.0 mg O2/L.
- When the oxygen concentration dropped below 2.0 mg O2/L, the measurement was stopped in accordance with the OECD guideline.
3 h EC50 > 1000 mg/L
NOEC = 206.6 mg/L

- The obtained results allowed to calculate the EC50 values on nitrification respiration. The results, calculated in terms of nominal test item concentrations, were as follows:
3 h EC50 = 903.4 mg/L

Results with reference substance (positive control):

Definitive test without ATU (total respiration RT):
3 h EC50= 10.52 mg/L

Definitive test with ATU (heterotrophic respiration RH):
3 h EC50: 11.36 mg/L

Reported statistics and error estimates:

The CETIS v.1.8.7.7. software was used to carry out the statistical analyses in order to assess the EC50 value and the NOEC value.
The EC50 calculation based on oxygen consumption rate was performed using a Linear Interpolation analysis for both test (without and with ATU).
The NOEC value was calculated by comparison with the negative control performed with Bonferroni Adj Test method for test without ATU and was performed with Wilcoxon/ Bonferroni Adj Test method for test with ATU.

The test meets the validity criteria described in OECD (N° 209, 2010), guideline:

- The negative controls specific respiration rate (R_s) mean value was equal to 30.5 and 24.4 mg O₂/g sludge/h after 3 hours, for test without and with ATU respectively, greater than the minimum value as required by the OECD guideline (N° 209, 2010), 20 mg oxygen per one gram of activated sludge (dry weight) in an hour.

-  The coefficient of variation in negative controls replicates was 7.0% and 12.7% after 3 hours results, for test without and with ATU respectively, thus complying with the validity criterion that provide a maximum coefficient of variation of 30% at the end of definitive test.

-  The EC₅₀value of 3,5-dichlorophenol was 10.52mg/L, in compliance with the validity range indicated by the reference guidelines (EC₅₀: 2 - 25 mg/L) for test without ATU (total respiration) and was 11.36 mg/L, in compliance with the validity range indicated by the reference guidelines (EC₅₀: 5 - 40 mg/L) for test with ATU (heterotrophic respiration).

Validity criteria fulfilled:

yes

Conclusions:

EC50 value for total respiration was assessed to be > 1000.0 mg test item/L and the NOEC value was 206.6 mg test item/L.
EC50 value for heterotrophic respiration was assessed to be > 1000.0 mg test item/L and the NOEC value was 454.5 mg test item/L.
EC50 value for nitrification respiration was assessed to be 903.4 mg test item/L.

Executive summary:

An OECD 209 compliant “Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)” was conducted to study the toxic effects of Sclareolide on aerobic treatment plant microorganisms under GLP conditions.

Since a preliminary test have shown a 60.8% of respiration inhibition at the highest concentration (1000 mg/L of test item), a definitive test at five concentrations was performed.

In addition, during the preliminary test, two additional blank bottles with N-allylthiourea (ATU) were performed, run in parallel to the blank bottles without ATU.

The difference between the respiration rate of the two series of blank bottles (without and with ATU), showed that the respiration rate due to the nitrification respiration is significant (> 5% of R_T), therefore two series of test bottles in definitive test were prepared, one series without ATU and one series with ATU.

This method permitted to measure the oxygen uptake due to the total respiration (R_T) and the oxygen uptake due to the heterotrophic respiration (R_H). The relationship between total respiration and heterotrophic respiration allows to calculate the oxygen uptake due to nitrification respiration (R_N).

EC₅₀ value for total respiration was assessed to be > 1000.0 mg/L and the NOEC value was 206.6 mg/L.

EC₅₀ value for heterotrophic respiration was assessed to be > 1000.0 mg/L and the NOEC value was 454.5 mg/L.

EC₅₀ value fornitrification respirationwas calculated to be 903.4mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

206.6 mg/L