Amines, C12-14-alkyl, C6-10-alkyl phosphates

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The pre-culture was prepared 5 days before the test start. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

The water-accommodated fraction (WAF) was prepared for the test. This was done by mix-ing 100.0 mg/L resp. 104.2 µL/L test item (based on a density of 0.96 g/mL stated in the SDS (given by the sponsor)) with the corresponding amount of algal medium (demineral-ised water enriched with minerals but without algae) and stirred moderately for 24 hours. The lower phase was used unfiltered as test solution. The lower treatments were prepared by dilution of this WAF with nutrient medium. The resulting solutions were used for preparation of the treatments. 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. In a non-GLP pre-test, at the loading rate of 1 mg/L, complete inhibition of algal growth was observed.

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
Taxonomic position Chlorophyta - Chlorophyceae
6.3.2 Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was pre-pared.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.0 – 23.4 °C

pH:

ca 7.9

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: "WAF", water accommodated fraction

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: "WAF", water accommodated fraction

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.52 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: "WAF", water accommodated fraction

Basis for effect:

growth rate

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. One valid experiment was performed. The study was performed using 6 concentrations ranging from 0.32 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 hours with an electronic particle counter. Growth rate µ and the yield[1] were determined from the cell number at the respective observation times. Significant inhibition of algal growth was observed at following concentrations:

1 – 100 mg/L (yield)

3.2 – 100 mg/L (growth rate)

The water-accommodated fraction (WAF) was stirred for 24 hours. The lower phase was used unfiltered as test solution. Lower test concentrations were prepared by dilution of the WAF in algal medium. 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. 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. In the 3 highest concentrated treatments the measured concentrations lay between 7 % and 19 % of the nominal concentrations at the beginning of the test and between 7 % and 14 % of the nominal concentrations at the end of the test. In the lower concentrations no test item was detectable. Since the lower concentrations were prepared by dilution of the stock WAF, the real concentrations were calculated on the basis of the measured concentration in the stock WAF and the dilution factor. Therefore the biological results were based on the nominal concentration and the calculated values. For the estimation of the EC50s of the test item, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The 72h-EC50s of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50s 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 0.4 units 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.

[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.

The following results for the test item Deophos 228 were determined:

Table 3‑a        Results of the test item (Nominal)

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	1.0 mg/L	3.2 mg/L	1.3 mg/L	3.6 mg/L
Yield	0.32 mg/L	1.0 mg/L	0.84 mg/L	1.8 mg/L

 

Table 3‑b        Results of the test item (calculated)

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	0.16 mg/L	0.52 mg/L	0.21 mg/L	0.58 mg/L
Yield	0.05 mg/L	0.16 mg/L	0.13 mg/L	0.29 mg/L

 

Validity criteria fulfilled:

yes

Conclusions:

Results of the test item (calculated)

Endpoint NOEC LOEC EC10 EC50
Growth Rate 0.16 mg/L 0.52 mg/L 0.21 mg/L 0.58 mg/L
Yield 0.05 mg/L 0.16 mg/L 0.13 mg/L 0.29 mg/L

Executive summary:

One valid experiment was performed. The study was performed using 6 concentrations ranging from 0.32 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 hours with an electronic particle counter. Growth rate µ and the yield[1]were determined from the cell number at the respective observation times. Significant inhibition of algal growth was observed at following concentrations: 1 – 100 mg/L (yield); 3.2 – 100 mg/L (growth rate) The water-accommodated fraction (WAF) was stirred for 24 hours. The lower phase was used unfiltered as test solution. Lower test concentrations were prepared by dilution of the WAF in algal medium. 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. 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. In the 3 highest concentrated treatments the measured concentrations lay between 7 % and 19 % of the nominal concentrations at the beginning of the test and between 7 % and 14 % of the nominal concentrations at the end of the test. In the lower concentrations no test item was detectable. Since the lower concentrations were prepared by dilution of the stock WAF, the real concentrations were calculated on the basis of the measured concentration in the stock WAF and the dilution factor.   Therefore the biological results were based on the nominal concentration and the calculated values.    The 72h-EC50s 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). 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. 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. The water-accommodated fraction (WAF) was stirred for 24 hours. The lower phase was used unfiltered as test solution. Lower test concentrations were prepared by dilution of the WAF in algal medium. 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. 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. In the 3 highest concentrated treatments the measured concentrations lay between 7 % and 19 % of the nominal concentrations at the beginning of the test and between 7 % and 14 % of the nominal concentrations at the end of the test. In the lower concentrations no test item was detectable. Since the lower concentrations were prepared by dilution of the stock WAF, the real concentrations were calculated on the basis of the measured concentration in the stock WAF and the dilution factor. Therefore the biological results were based on the nominal concentration and the calculated values.

Under the study conditions, the 72 h NOEC and ErC50 for growth rate were determined to be 0.16 mg/L and 0.58 mg/L respectively. The 72 h NOEC and EbC50 for yield were determined to be 0.05 mg/L and 0.29 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.58 mg/L

EC10 or NOEC for freshwater algae:

0.16 mg/L

Additional information

One valid experiment was performed.The study was performed using 6 concentrations ranging from 0.32 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.Significant inhibition of algal growth was observed at following concentrations: 1 – 100 mg/L (yield);3.2 – 100 mg/L (growth rate)The water-accommodated fraction (WAF) was stirred for 24 hours. The lower phase was used unfiltered as test solution. Lower test concentrations were prepared by dilution of the WAF in algal medium. 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.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.In the 3 highest concentrated treatments the measured concentrations lay between 7 % and 19 % of the nominal concentrations at the beginning of the test and between 7 % and 14 % of the nominal concentrations at the end of the test. In the lower concentrations no test item was detectable.Since the lower concentrations were prepared by dilution of the stock WAF, the real concentrations were calculated on the basis of the measured concentration in the stock WAF and the dilution factor. Therefore the biological results were based on the nominal concentration and the calculated values.  The 72h-EC50s 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). 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. 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. The water-accommodated fraction (WAF) was stirred for 24 hours. The lower phase was used unfiltered as test solution. Lower test concentrations were prepared by dilution of the WAF in algal medium. 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. 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. In the 3 highest concentrated treatments the measured concentrations lay between 7 % and 19 % of the nominal concentrations at the beginning of the test and between 7 % and 14 % of the nominal concentrations at the end of the test. In the lower concentrations no test item was detectable. Since the lower concentrations were prepared by dilution of the stock WAF, the real concentrations were calculated on the basis of the measured concentration in the stock WAF and the dilution factor. Therefore the biological results were based on the nominal concentration and the calculated values.

Under the study conditions, the 72 h NOEC and ErC50 for growth rate were determined to be 0.16 mg/L and 0.58 mg/L respectively. The 72 h NOEC and EbC50 for yield were determined to be 0.05 mg/L and 0.29 mg/L respectively.