Phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2'-iminobis[ethanol]

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

Study period:

From December 09, 2016 to February 08, 2017

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 92.75 %; Appearance: lightly yellow paste; Solubility in water: 600 mg/L; Stability in water: stable

Analytical monitoring:

yes

Details on sampling:

Due to the complex nature of the test substance, specific analyses proved to be difficult to obtain, therefore it was decided to analyse the Total Organic Carbon (TOC) concentration as a surrogate measure.
Single samples for possible analysis were taken from all test groups without algae according to the schedule below.
Frequency: at t=0 h, and t=72 h
Volume: 40 mL
Storage Samples: were stored in a refrigerator until analysis.

Additionally, single reserve samples of 40 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a refrigerator for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

Preliminary data: the TOC content of the test substance was measured to be 56.85 %.

The batch of the test substance was a lightly yellow paste with a combined purity of 92.75 % and not completely soluble in test medium at the concentrations tested. A correction was made for the purity of the test substance. A correction factor of 1.07 was used. Concentrations are subsequently expressed based on the active ingredients.
Test solutions were prepared separately at loading rates ranging from 1.1 to 107 mg/L, applying a two-day period of magnetic stirring after which the resulting mixtures were allowed to stabilize overnight. Subsequently, the Water Accommodated Fractions (WAFs) were siphoned off over glass wool. WAFs prepared at 19 mg/L and less were clear and colorless, while WAFs prepared at higher loading rates were found to be still colorless but increasingly turbid with increasing loading rate.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Combined limit/range-finding test: WAF prepared at a loading rate of 107 mg/L.
Test concentrations in the final test: WAFs prepared at loading rates of 11, 19, 34, 60 and 107 mg/L.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1,
Source: in-house laboratory culture,
Reason for selection: this system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Pre-culture medium M2: Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L).

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 24°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

pH:

The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 units).

Nominal and measured concentrations:

Combined limit/range-finding test: WAF prepared at a loading rate ranging from 1.1 to 107 mg/L.
Test concentrations in the final test: WAFs prepared at loading rates of: 11, 19, 34, 60 and 107 mg/L.
Measured concentrations final test:
Samples for TOC concentrations were taken at the start and after 72 hours of exposure. The measured TOC concentrations remained stable throughout the test duration i.e. were at 39 to 52 % of the nominal loading rates at the begging and end of the test.

Details on test conditions:

Fresh water algae culture

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24 °C.

Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium M1: according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedfo Mass., USA).

Pre-culture 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 19-26 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

101 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 93-112 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CI 10-14 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

34 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95% CI 32-37 mg/L

Details on results:

Combined limit/range-finding test

Results
Significant effects on algal growth rate and yield were observed in the highest test group while effects in the treatments with nominal loading rates of 11 mg/L and less were negligible. The expected EL50 for growth rate inhibition was beyond the tested concentration range, while the expected EL50 for yield inhibition ranged between nominal 11 and 107 mg/L.

Measured test substance concentrations - range findng test
Samples taken from nominal 107 mg/L were analysed. The initial TOC concentration was 20 mg/L, corresponding to 33% of the nominal TOC concentration as based on the loading rate. This concentration remained stable during the test period, i.e., was 104 % of initial after 72 hours.


Final tast

Results:
Growth rates were in the range of the controls at the two lowest test concentrations. Inhibition of growth rate was statistically significant at nominal loading rates of 34 mg/L and higher. Inhibition of yield resulted in 93 % reduction at the highest test concentration. Statistically significant inhibition of yield was found at nominal loading rates of 34 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of algal cells from all test groups when compared to the control.
The EL50 for growth rate inhibition (72h-ERL50) was 108 mg/L with a 95 % confidence interval ranging from 99 to 120 mg/L.
The EL50 for yield inhibition (72h-EYL50) was 37 mg/L with a 95 % confidence interval ranging from 34 to 40 mg/L.
The 72h-NOEL for both growth rate inhibition and yield inhibition were 19 mg/L.

Measured test substance concentrations - final test:
Samples for TOC concentrations were taken at the start and after 72 hours of exposure. The measured TOC concentrations remained stable throughout the test duration i.e. were at 39 to 52 % of the nominal loading rates at the begining and end of the test. Since TOC-analysis is a non-specific method, results are reported in terms of the nominal loading rates.

Results with reference substance (positive control):

Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.32 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95 % confidence interval ranging from 1.2 to 1.4 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.45 mg/L with a 95 % confidence interval ranging from 0.39 to 0.53 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Determination of the NOEL and calculation of the EL50

For determination of the NOEL and the EL50, the approaches recommended in the OECD Guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Additionally, the EL10 and EL20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ELx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal loading rate of the test item.
At the end of the test duration, replicate 1 of the test group prepared at 34 mg/L was judged to
be an outlier since the determined cell density appeared uncharacteristically high. The replicate was consequently removed from the statistical analysis of the final test. The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions®
GmbH, Germany).

Acceptability of the test
1. In the controls, cell density increased by an average factor of at least 16 within the exposure period (i.e. 107).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 19%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 2.8%).

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the nominal EL50 of the substance in the aquatic algae for growth rate and yield inhibition were determined to be 101 mg/L and 34 mg/L respectively.

Executive summary:

A study was conducted to determine the toxicity of the substance to Pseudokirchneriella subcapitata according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Test solutions were prepared separately at loading rates ranging from 1.1 to 107 mg/L, applying a two-day period of magnetic stirring after which the resulting mixtures were allowed to stabilize overnight. Subsequently, the Water Accommodated Fractions (WAFs) were siphoned off over glass wool. WAFs prepared at 19 mg/L and less were clear and colorless, while WAFs prepared at higher loading rates were found to be colorless but increasingly turbid with increasing loading rate. A final test was performed based on the results of a preliminary combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at 11, 19, 34, 60 and 107 mg/L. The initial algal cell density was 10E+4 cells/mL. The total exposure period was 72 h. Due to the complex nature of the test substance, specific analyses proved to be difficult to obtain and, therefore it was decided to analyse the Total Organic Carbon (TOC) concentration as a surrogate measure. Samples for TOC concentrations were taken at the start and after 72 h of exposure. The measured TOC concentrations remained stable throughout the test duration i.e. were at 39 to 52 % of the nominal loading rates at the begging and end of the test. Since TOC-analysis is a non-specific method, results are reported in terms of the nominal loading rates. The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the study conditions, the nominal EL50 of the substance in the aquatic algae for growth rate and yield inhibition were determined to be 101 mg/L and 34 mg/L respectively (Augusiak, 2017).

Key value for chemical safety assessment

EC50 for freshwater algae:

101 mg/L

Additional information

A study was conducted to determine the toxicity of the substance to Pseudokirchneriella subcapitata according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Test solutions were prepared separately at loading rates ranging from 1.1 to 107 mg/L, applying a two-day period of magnetic stirring after which the resulting mixtures were allowed to stabilize overnight. Subsequently, the Water Accommodated Fractions (WAFs) were siphoned off over glass wool. WAFs prepared at 19 mg/L and less were clear and colorless, while WAFs prepared at higher loading rates were found to be colorless but increasingly turbid with increasing loading rate. A final test was performed based on the results of a preliminary combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at 11, 19, 34, 60 and 107 mg/L. The initial algal cell density was 10E+4 cells/mL. The total exposure period was 72 h. Due to the complex nature of the test substance, specific analyses proved to be difficult to obtain and, therefore it was decided to analyse the Total Organic Carbon (TOC) concentration as a surrogate measure. Samples for TOC concentrations were taken at the start and after 72 h of exposure. The measured TOC concentrations remained stable throughout the test duration i.e. were at 39 to 52 % of the nominal loading rates at the begging and end of the test. Since TOC-analysis is a non-specific method, results are reported in terms of the nominal loading rates. The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the study conditions, the nominal EL50 of the substance in the aquatic algae for growth rate and yield inhibition were determined to be 101 mg/L and 34 mg/L respectively (Augusiak, 2017).