3,5-bis(2,4-dimethylcyclohex-3-en-1-yl)polyheterocycle

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 - 20 - 2017 -- 06 - 08 - 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Incubation: 3 hours

Test solutions

Vehicle:

no

Details on test solutions:

Stock Solution of 3,5-Dichlorophenol: The reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 1, 4 and 16 mg/L (five replicates for each test concentration) under otherwise identical test conditions.
A stock solution of 3,5-dichlorophenol was prepared according to the OECD Guideline No. 209: 250 mg of 3,5-dichloro-phenol was dissolved in 250 mL pure water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it had cooled to room temperature. The final pH was 7.1 and therefore in the range of 7 to 8. NaOH was used for the adjustment of the pH.
Stock Solution of N-allylthiourea (ATU): In parallel a nitrification inhibitor N-allylthiourea (ATU) was tested in the same way with six separate controls and at the identical nominal concentrations of the test item and also the reference item 3,5-dichlorophenol under otherwise identical test conditions.
A stock solution of N-allylthiourea was prepared according to the OECD Guideline No. 209: a stock solution of 2.32 g/L N-allylthiourea was prepared. 2.5 mL of this stock solution were added to an incubation mixture of final volume of 500 mL. This resulted in a final concentration of 11.6 mg ATU/L.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Species / Origin: Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.
Conditioning: The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed with 50 mL synthetic sewage feed (see below) per litre and kept aerated at room temperature overnight.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gives a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge inoculum was 6.8 and therefore no adjustment necessary.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

For the measurement of the respiration rate, a sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes.

Test conditions

Test temperature:

20°C ± 2°C during pre-incubation (3 hours) and evaluation period

pH:

7.0 - 8.0

Dissolved oxygen:

8.9 to 0.8 mg O2/L for total respiration

Nominal and measured concentrations:

Test Concentrations:
Nominal 10, 32, 100, 320 and 1000 mg test item/L
1, 4 and 16 mg 3,5-Dichlorphenol/L and six inoculum controls

Details on test conditions:

Controlled environment room
Temperature: 20°C ± 2°C during pre-incubation (3 hours) and evaluation period
Aeration: Compressed air (1.017 litre per minute)

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Inhibition of Total Respiration Rate:

In comparison to the inoculum controls the total respiration rate of the activated sludge was not inhibited by the two lower test concentrations and only slightly inhibited for the other test concentrations. The inhibition was 8.6% at a test concentration of 10 mg/L and 4.4% at a test item concentration of 32 mg/L. For the test item concentrations of 100 mg/L and 320 mg/L the inhibition rates were 12.6%, and 17.03%, respectively. The highest test concentration of 1000 mg/L resulted in an inhibition of 16.3%. Concentrations exceeding 1000 mg/L nominal were not tested.
The respiration rates of test item concentrations of 10 mg/L and 32 mg/L were not significantly different from the control. Test item concentrations of 100 mg/L320 mg/L and 1000 mg/L showed significant differences compared to control. A slight concentration related inhibiting effect could be determined for test item concentrations above 32 mg/L.

3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Total Respiration:

As the inhibition was below 20% for all tested concentrations, no 3-hour EC50 for total respiration could be established. The 3-hour EC20 was defined to be above the highest tested concentration of 1000 mg/L (1666 mg/L), the EC10 was calculated to be 48 mg/L.
The NOEC was determined to be at a test item concentration of 32 mg/L.

The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/l] 48.1 1666.2 n.d.
lower 95%-cl n.d. n.d. n.d.
upper 95%-cl n.d. n.d. n.d.
n.d.: not determined due to mathematical reasons

Inhibition of Heterotrophic Respiration Rate:
In comparison to the inoculum controls, the heterotrophic respiration rate was constantly inhibited between 22% and 28% for all test item concentrations. The inhibition rates were 22.0%, 23.0%, 27.3%, 26.8% and 28.3% for test item concentrations of 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L and 1000 mg/L, respectively.

The respiration rates of all test item concentrations were significantly different from the control. A concentration related inhibiting effect could not be determined; therefore the effect seems not to be a clear effect of the test item.


3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Heterotrophic Respiration:
As no concentration-related inhibition of the heterotrophic respiration was determined, no 3-hour ECx values could be calculated with statistical procedures.

 
Inhibition of Nitrification Respiration Rate:

In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification was not inhibited by test item concentrations between 10 mg/L and 1000 mg/L (inhibition below 5% for all test item treatments).
The respiration rates of all test item concentrations were not significantly different from the control.


3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Nitrification Respiration:
No ECx values were determined; the NOEC was determined to be above a test item concentration of 1000 mg/L.

Results with reference substance (positive control):

Toxicity of 3,5-Dichlorophenol: The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration was found to be 5.7 mg/L thus lying in the range of 2 - 25 mg/L recommended by the test guidelines.
The 3-hour EC50 for heterotrophic respiration was found to be 15.2 mg/L thus lying in the range of 5 - 40 mg/L recommended by the test guidelines.
For the oxygen uptake due to nitrification the 3-hour EC50 was 1.1 mg/L, lying in the range of 0.1 - 10 mg/L recommended by the test guidelines.
The result confirms the suitability of the activated sludge used.

Reported statistics and error estimates:

Statistical Analysis:

Test item

Total
Heterotroph
Nitrification
ECx Estimation:



Regression Analysis:
3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting;
Optimization method: Levenberg-Marquardt
3-Parametric normal CDF R2:
Terminated without achieving convergence due to mathematical problems
0.974 (a significant amount of variance is explained by the regression model)
0.089 (the amount of variance explained by the model is not significant)
NOEC estimation:



Test on Normal Distribution:
Shapiro Wilk’s test (α= 0.01)
Variance Homogeneity:
Levene’s Test (α= 0.01)
T-test Procedure for treatment comparison and NOEC estimation:
Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm
(α= 0.05, one-sided smaller)
Williams Multiple Sequential t-test
(α= 0.05, one-sided smaller)
Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm;
(α= 0.05, one-sided smaller)


Reference item

Total
Heterotroph
Nitrification
ECx Estimation:



Regression Analysis:
3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting;
Optimization method: Levenberg-Marquardt
3-Parametric normal CDF R2:
0.944
0.996
0.904

The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ® ToxRat Solutions GmbH.

Any other information on results incl. tables

Influence of Sa 57 and the Reference Item 3,5-Dichlorophenol on the Total Respiration Rate of Aerobic Waste Water Microorganisms after 3 Hours of Exposure

Flask No.	Treatment	Concen-tration	Oxygen consumption^1	Standard Deviation^1	Oxygen consumption^3	Inhibition^1	pH-values^2		Oxygen concentration [mg O2/L]^2
		[mg/L]	[mg O2/(L*h)]	%	[mg O2/(g*h)]	%	start*	end*	start*	end*
1, 2, 18, 19, 45,46	control	---	39.4	3.5	26.3	---	6.9	7.7	6.7	7.0
3-7	3.5-DCP	1	31.2	6.9	20.8	20.8	7.0	7.9	6.8	7.2
8-12	3.5-DCP	4	25.0	2.8	16.6	36.6	6.9	7.7	6.6	8.3
13-17	3.5-DCP	16	8.9	3.8	5.9	77.5	6.7	7.3	6.6	8.7
20-24	Test item	10	36.0	2.4	24.0	8.6	6.9	7.8	6.7	7.3
25-29	Test item	32	37.7	2.1	25.1	4.4	7.0	7.1	6.8	6.1
30-34	Test item	100	34.4	3.3	22.9	12.6	7.0	7.1	6.9	6.5
35-39	Test item	320	32.6	2.0	21.7	17.3	6.9	7.5	6.6	5.9
40-44	Test item	1000	33.0	4.3	22.0	16.3	6.8	7.2	7.0	6.9
CV of control [%]:		8.9
^1: mean value of 5 replicates, 6 replicates in case of control ^2: mean value of 3 replicates for control; measured in replicate 1 for 3,5-DCP and test item ^3: calculated from the mean oxygen consumption in mg O2/(L*h) per treatment and the used sludge concentration of 1.5 g/L *: Start and end of 3-hour aeration DCP: 3,5-Dichlorophenol Test item: 1H, 3H, 5H-Oxazolo[3,4-c]oxazole, 3,5-bis(2,4-dimethyl-3-cyclohexen-1-yl) dihydro- CV: standard d eviation / mean * 100

Influence of Sa 57 and the Reference Item 3,5-Dichlorophenolon the Heterotrophic Respiration Rate of Aerobic Waste Water Microorganisms after 3 Hours of Exposure

Flask No.	Treatment	Concen-tration	Oxygen consumption^1	Standard Deviation^1	Oxygen consumption^3	Inhibition^1	pH-values^2		Oxygen concentration [mg O2/L]^2
		[mg/L]	[mg O2/(L* h)]	%	[mg O2/(g* h)]	%	start*	end*	start*	end*
47, 48, 64, 65, 91, 92	control	---	22.6	1.1	15.1	---	6.9	7.8	6.7	7.6
49-53	3.5-DCP	1	22.1	5.4	14.7	2.3	7.1	7.8	6.7	7.3
54-58	3.5-DCP	4	23.3	10.9	15.5	-3.0	6.9	7.9	6.7	7.8
59-63	3.5-DCP	16	7.4	3.6	5.0	67.1	6.7	7.3	6.8	8.4
66-70	Test item	10	17.6	3.6	11.8	22.0	6.9	7.8	6.8	7.9
71-75	Test item	32	17.4	4.4	11.6	23.0	6.9	7.9	6.4	7.6
76-80	Test item	100	16.4	3.6	11.0	27.3	6.9	7.8	6.8	7.6
81-85	Test item	320	16.6	4.0	11.0	26.8	6.9	7.8	7.0	7.4
86-90	Test item	1000	16.2	3.7	10.8	28.3	6.8	7.9	6.9	7.7
CV of control [%]:		4.9
^1: mean value of 5 replicates, 6 replicates in case of control ^2: mean value of 3 replicates for control; measured in replicate 1 for 3,5-DCP and test item ^3: calculated from the mean oxygen consumption in mg O2/(L*h) per treatment and the used sludge concentration of 1.5 g/L *: Start and end of 3-hour aeration DCP: 3,5-Dichlorophenol Test item: 1H, 3H, 5H-Oxazolo[3,4-c]oxazole, 3,5-bis(2,4-dimethyl-3-cyclohexen-1-yl) dihydro-

Influence of 1H, 3H, 5H-Oxazolo[3,4-c]oxazole, 3,5-bis(2,4-dimethyl-3-cyclohexen-1-yl) dihydro- and the Reference Item 3,5-Dichlorophenol on the Nitrification RespirationRate of Aerobic Waste Water Microorganisms after 3 Hours of Exposure

Treatment	Concentration [mg/L]	Oxygen consumption^1[mg O2/(L*h)]	Standard Deviation^1   %	Inhibition^1%
control	---	16.8	3.2	---
3.5-DCP	1	9.1	16.3	45.7
3.5-DCP	4	1.7	6.6	90.0
3.5-DCP	16	1.4	8.9	91.4
Test item	10	18.4	5.7	-9.4
Test item	32	20.3	4.9	-20.7
Test item	100	18.0	7.7	-7.0
Test item	320	16.0	4.7	4.6
Test item	1000	16.8	10.1	0.2
^1: mean value of 5 replicates, 6 replicates in case of control *: Start and end of 3-hour aeration DCP: 3,5-Dichlorophenol Test item: 1H, 3H, 5H-Oxazolo[3,4-c]oxazole, 3,5-bis(2,4-dimethyl-3-cyclohexen-1-yl) dihydro-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Sa 57 had slightly inhibiting effects on total respiration rate for concentrations of 100 mg/L and higher. A slight concentration related inhibiting effect could be determined for test item concentrations above 32 mg/L.
Sa 57 had no concentration-related inhibiting effect on heterotrophic respiration rate. The slightly inhibiting effects over the tested concentration range could not clearly be attributed to the test item.
The nitrification respiration was not inhibited for the tested concentrations up to and including 1000 mg/L.



Therefore, it is concluded that the test item had slightly inhibiting effects on total respiration, resulting with a NOEC of 32 mg/L. No EC50 could be determined.

Executive summary:

Title:	Sa 57: Toxicity to Activated Sludge in a Respiration Inhibition Test
Guidelines/Recommendations:	This study is designed to comply with the following methods: -     Commission Regulation (EC) No 440/2008, Method C.11: "Activated Sludge Respiration Inhibition Test", Official Journal of the European Union No. L 142/559-563, dated May 30, 2008 -     OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)", adopted July 22, 2010 -     Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation (ISO 8192:2007); German version EN ISO 8192:2007
Purpose:	The influence of the test item Sa 57 on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage feed was measured in the presence of various concentrations of the test item after an incubation period of 3 hours.
Test Concentrations:	10, 32, 100, 320 and 1000 mg /L Sa 57; 1, 4 and 16 mg 3,5-Dichlorophenol/L and Six inoculum controls
Results:
Toxicity of Test Item:	The test item Sa 57 was weighed dosed into each test flask and pure water was added. The test item was dissolved into the pure water as homogeneously as possible.   Total respiration: In comparison to the inoculum controls the total respiration rate of the activated sludge was not inhibited at the two lower test concentrations and only slightly inhibited for the other test concentrations. The inhibition was 8.6% at a test concentration of 10 mg/L and 4.4% at a test item concentration of 32 mg/L. For the test item concentrations of 100 mg/L and 320 mg/L the inhibition rates were 12.6%, and 17.03%, respectively. The highest test concentration of 1000 mg/L resulted in an inhibition of 16.3%. Concentrations exceeding 1000 mg/L nominal were not tested. A slight concentration related inhibiting effect could be determined for test item concentrations above 32 mg/L. As the inhibition was below 20% for all tested concentrations, no 3-hour EC50for total respiration could be established. The 3-hour EC20was defined to be above the highest tested concentration of 1000 mg/L (1666 mg/L), the EC10was calculated to be 48 mg/L. The NOEC was determined to be at a test item concentration of 32 mg/L.    Heterotrophic Respiration (Respiration without nitrification ): In comparison to the inoculum controls the heterotroph respiration was constantly inhibited between 22% and 28% for all test item concentrations. The inhibition rates were 22.0%, 23.0%, 27.3%, 26.8% and 28.3% for test item concentrations of 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L and 1000 mg/L, respectively. A concentration related inhibiting effect could not be determined; therefore the effect seems not to be a clear effect of the test item. No 3-hour ECxvalues could be calculated with statistical procedures, as no concentration-related inhibition of the heterotrophic respiration was determined.   Respiration based on nitrification: In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification was not inhibited by test item concentrations between 10 mg/L and 1000 mg/L (inhibition below 5% for all test item treatments). As a consequence, the NOEC was determined to be above a test item concentration of 1000 mg/L.
Conclusion:	Sa 57 had slightly inhibiting effects on total respiration rate for concentrations of 100 mg/L and higher. A slight concentration related inhibiting effect could be determined for test item concentrations above 32 mg/L. Sa 57 had no concentration-related inhibiting effect on heterotrophic respiration rate. The slightly inhibiting effects over the tested concentration range could not clearly be attributed to the test item. The nitrification respiration was not inhibited for the tested concentrations up to and including 1000 mg/L. Therefore, it is concluded that the test item Sa 57 had slightly inhibiting effects on total respiration, resulting with a NOEC of 32 mg/L. No EC50could be determined.
Toxicity of 3,5-Dichlorophenol:	The positive control 3,5-Dichlorophenol was tested in the same way as the test item.The 3-hour EC50for total respiration was found to be 5.7 mg/Lthus lying in the range of 2 - 25 mg/L recommended by the test guidelines. The 3-hour EC50for heterotrophic respiration was found to be 15.2 mg/L thus lying in the range of 5 - 40 mg/L recommended by the test guidelines. For the oxygen uptake due to nitrification the 3-hour EC50was 1.1 mg/L, lying in the range of 0.1 - 10 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used.