Terpene Dimers

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2018- December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Deviations:
The following deviations were documented:
♦ The temperature in Experiment 3 was in the range 21.7 – 24.2°C, and therefore not in the desired range (21.0 - 24.0°C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
♦ The temperature during pre-culture incubation (Experiment 1) was in the range 19.4 – 22.7°C, and therefore not in the desired range (21.0 - 24.0°C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
♦ The temperature during pre-culture incubation (Experiment 2) was in the range 20.9
– 22.0°C, and therefore not in the desired range (21.0 - 24.0°C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

Deviations:

yes

Remarks:

see Version/remarks

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Terpene Dimers

EC Number:

948-032-2

IUPAC Name:

Terpene Dimers

Test material form:

liquid

Details on test material:

Batch no. DF1710011

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

Experiment 1 and 2:
The water-accommodated fractions (WAF) were prepared for the test. This was done by mixing the nominal loads of 1 / 3.2 / 10 / 32 / 100 mg/L ± 1 mg/L resp. 1.05 / 3.36 / 10.5 / 33.6 / 105 ± 1 µL/L test item (based on a density of 0.9526 g/cm3 given by the sponsor with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring moderate for 7 days at 130 rpm. After a settling phase of about 24 ± 0.5 hours the lower phase was used unfiltered for the test.

Experiment 3:
The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing the nominal load of 100.0 mg/L resp. 105 µL/L test item (based on a density of 0.9526 g/cm3 given by the sponsor) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring moderate for 7 days at 130 rpm. After a settling phase of about 24.5 hours the lower phase was used unfiltered for the test. The lower treatments were prepared by dilution of this WAF with algal medium. This procedure is in agreement with the OECD guidance document no. 23 for the testing of mixtures which are toxic at very low loading rates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test conditions

Test temperature:

21.8 – 23.7 °C (Experiment 1)
22.0 – 23.3 °C (Experiment 2)
21.7 – 24.2 °C (Experiment 3)

pH:

7.7 -9.1

Nominal and measured concentrations:

nominal loads of 1 / 3.2 / 10 / 32 / 100 mg/L ± 1 mg/L resp.

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser. Since the analytical results are all in the same very low range as the blank value (< 1 mg/L TOC), the results are related to the nominal values. However, these results can show that the test item is present in the solution.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

75.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

49.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Three experiments were performed.
The results of the first and second experiment are stated in the report. The statistic evaluation was only conducted with the results of the third experiment.

The studies were performed using 5 concentrations ranging from 1.0 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 ± 1 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

Experiment 1:
The inhibition values were not in agreement with the different loading rates and behaved differently than could be expected from the pre-test. The analytical results also varied and were not conclusive. Therefore a second experiment was performed in the same way.

Experiment 2:
In the second experiment, similar inhibition values occurred in all treatments. This time, however, the values were comparable to those of the pre-test. This suggests that the same amount of test item was dissolved in all treatments. Again, very low DOC values had been measured.
Therefore no NOEC/LOEC-values (resp. NOELR/LOELR-values) could be determined.
Since the lowest concentration showed a clear inhibition of algae growth, the test preparation was adapted in the third experiment. A stock WAF (water-accommodated fraction) was prepared and diluted. This was in accordance OECD Guidance Document No. 23 where is stated that serial dilution of a stock WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.

Experiment 3:
In the third experiment a clear dose-response-relationship was observed.
Significant inhibition of algal growth could be observed in the following concentrations: 10 – 100 mg/L (nominal) for growth rate and yield

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.
Since the analytical results are all in the same very low range as the blank value (< 1 mg/L TOC), the results are related to the nominal values. However, these results indicate that the test item is present in the solution.
The inhibition values related to the growth rate do not reach 50%, and therefore no EC50 resp. EL50 value could be calculated. Since the test item is an insoluble UVCB, this value cannot be extrapolated. Therefore it is given as a range. Since some of the effect values refer to the loading rate, in this case NOELR (No Observable Effect Loading Rate) or EL50values are used.

The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.

The pH of the blank control should not fluctuate by more than 1.5 units.
The change was 1.3 units (Experiment 1), 0.3 units (Experiment 2) and respectively 0.2 units (Experiment 3) in the blank control.

All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Executive summary:

Findings and Results:

 

Three experiments were performed. 

The results of the first and second experiment are stated in the report. The statistic evaluation was only conducted with the results of the third experiment.

 

The studies were performed using 5 concentrations ranging from 1.0 to 100 mg/L. Incubation time (test systemDesmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield^[1]were determined from the cell number at the respective observation times.

 

Experiment 1

The inhibition values were not in agreement with the different loading rates and behaved differently than could be expected from the pre-test. The analytical results also varied and were not conclusive. Therefore a second experiment was performed in the same way.

 

Experiment 2

In the second experiment, similar inhibition values occurred in all treatments. This time, however, the values were comparable to those of the pre-test. This suggests that the same amount of test item was dissolved in all treatments. Again, very low DOC values had been measured.

Therefore no NOEC/LOEC-values (resp.NOELR/LOELR-values) could be determined. 

Since the lowest concentration showed a clear inhibition of algae growth, the test preparation was adapted in the third experiment. A stock WAF (water-accommodated fraction) was prepared and diluted. This was in accordance with OECD Guidance Document No. 23 where is stated that serial dilution of a stock WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.

 

Experiment 3

In the third experiment a clear dose-response-relationship was observed. 

Significant inhibition of algal growth could be observed in the following concentrations:

10 – 100 mg/L (nominal) for growth rate and yield 

 

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser. 

Since the analytical results are all in the same very low range as the blank value (< 1 mg/L TOC), the results are related to the nominal values. However, the clear dose-responserelationship indicate that the test item is present in the test solution.

The inhibition values related to the growth rate do not reach 50%, and therefore no EC₅₀resp.EL₅₀ value could be calculated.Since the test item is a poor soluble UVCB, this value cannot be extrapolated. Therefore it is given as a range. Since some of the effect values refer to the loading rate, in this case NOELR (No Observable Effect Loading Rate) or EL₅₀-values are used.

 

The 72h-EC₅₀values of potassium dichromate (K₂Cr₂O₇, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item Terpene Dimers were determined:

Table 3-a        Results of the test item

Endpoint	NOEC / NOELR	LOEC / LOELR	EC10/ EL10	EC50/ EL50
Growth Rate	3.20 mg/L	10.00 mg/L	49.57 mg/L	> 100 mg/L
Yield	3.20 mg/L	10.00 mg/L	13.80 mg/L	75.18 mg/L

[1]Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period. Calculation see under 8.2 Growth and growth inhibition are quantified from measurements of the algal biomass as a function of time. Algal biomass is defined as the dry weight per volume, e.g. mg algae/L test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used.