Alcohols, C16-18 and ethoxylated C16-18 , phosphates (20 moles ethoxylation)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 30, 2017 to November 03, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Liquid chromatography mass spectrometry (LC/MS) method

Details on sampling:

To demonstrate that test vessels were dosed with nominal exposure concentrations of test substance, whole test vessels were analysed for test substance using the liquid chromatography mass spectrometry method.

Vehicle:

yes

Remarks:

AAP medium

Details on test solutions:

Preparation of test solutions
The study was run with a culture medium control and nominal exposure concentrations of 0.097, 2.13, 4.7, 10.3 and 23 mg/L. The nominal 0.097 mg/L test concentration was set up in error instead of 0.97 mg/L. As there was no significant effect at this concentration it was deemed not to affect the validity of the study. A primary stock concentrate of Test substance, with a nominal concentration of 50 mg/L, was prepared by adding a nominal 0.05 g of test substance (actual weight: 0.04981 g) to 1000 mL of media. The resultant stock was observed to be a colourless hazy solution with small particles visible and bubbles on the surface. The stock solution was used to prepare the test solutions. This was achieved by the direct addition of the appropriate amount of concentrate to dilution water. The control consisted of culture medium only. In all cases the final solutions contained nutrients. The dilution water control and 0.097 mg/L test solutions were observed to be clear and colourless. The 2.13 and 4.7 mg/L test solutions were observed to be colourless with small particles visible. The 10.3 and 23 mg/L test solutions were observed to be slightly hazy and hazy colourless solutions respectively, both with small particles visible and bubbles on the surface. The nominal 2.13, 4.7, 10.3 and 23 mg/L test solutions are therefore classified as homogeneous dispersions. The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.

Test organisms (species):

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

Details on test organisms:

The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 3-d old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium and under the environmental conditions described for the test. The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium.

Test type:

not specified

Water media type:

other: AAP-medium

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2ºC

pH:

6.95 to 8.15

Nominal and measured concentrations:

23 mg/L test concentration was selected as the highest concentration based on the toxicity demonstrated in a non-GLP range-finding study.
0, 0.097a, 2.13, 4.7, 10.3 and 23 mg/L (Nominal)
0, 1.5, 3.8, 8.5 and 19 mg/L (Mean measured)
a The nominal 0.097 mg/L test concentration was set up in error instead of 0.97 mg/L. As there was no significant effect at this concentration it was deemed not to affect the validity of the study.

Details on test conditions:

Appratus
The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 22  2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking of approximately 160 rpm.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 19 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

ca. 3.15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 3.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: growth rate and biomass

Results:

Analytical data

The limit of quantification of Test substance in this study was 0.6 mg/L for all test solutions with the exception of the nominal 23 mg/L which was 1.8 mg/L. All analytical values are quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the study were 0-90% of nominal and at the end were 0-80% of nominal based on whole vessel extraction.The nominal 0.097 mg/L test solution was below the LOQ and as such was not detected at either time point. This concentration was below the NOEC and as such not having a measured analytical value does not impact the result of the study.Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R²value of 0.99. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 30% and less than 20% at levels greater than the LOQ.On the basis of the analytical data the mean measuredconcentrations were used for the calculation and reporting of results.

Biological data

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass.

Growth rates

The growth rate (0 to 72 h) was calculated for each replicate culture. The growth rates were examined by one-way analysis of variance and a Bonferroni adjusted t test to identify significant differences (p<0.05) from the control. The EC₅₀, EC₂₀ and EC₁₀ values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method (ICPIN).

The mean growth rates, are given in below table together with the rates expressed as percentages of the control.

Nominal concentration of test substance (mg/L)	Mean measured concentration of Test substance (mg/L)	Mean growth rate/day (0-72 h)	Mean growth rate/day 95% Cl	Percentage ofcontrol (%)
Culture medium control	0	1.501	1.476-1.527	-
0.097	0	1.498	1.471-1.526	100
2.13	1.5	1.473	1.436-1.510	98
4.7	3.8	1.142 *	0.9988-1.286	76
10.3	8.5	0.971 *	0.886-1.055	65
23	19	0.787 *	0.674-0.900	52

* Significant differences (p <0.05) from the control

All biological measurements are quoted to 3 decimal places and percentages to the nearest integer.

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)	95% confidence limits (mg/L)
NOEC	1.5	-
LOEC	3.8	-
ErC50	>19	N/A
ErC20	3.39	2.88 - 4.50
ErC10	2.35	2.00 - 2.83

Yield

The response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. These cell particle densities were examined by one-way analysis of variance and a Bonferroni adjusted t test to identify significant differences (p<0.05) from the control.The EC₅₀, EC₂₀ and EC₁₀ values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method (ICPIN).

The yield mean values are shown in below table, together with the yields expressed as percentages of the control.

Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Mean yield (0-72 h) (10^4cells/ml)	Mean yield 95% Cl	Percentage ofcontrol (%)
Culture medium control	0	42.5	39.2-45.9	-
0.097	0	42.1	38.5-45.6	99
2.13	1.5	39.0	34.6-43.4	92
4.7	3.8	14.3 *	7.76-20.9	34
10.3	8.5	8.28 *	6.13-10.4	19
23	19	4.59 *	2.91-6.27	11

* Significant differences (p <0.05) from the control

Mean yield values are quoted to 3 significant figures and percentages to the nearest integer.

The results obtained from these statistical analysis, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)	95% confidence limits (mg/L)
NOEC	1.5	-
LOEC	3.8	-
EyC50	3.15	2.79 - 3.67
EyC20	1.97	1.51 - 2.39
EyC10	1.57	0.481 - 2.5

Additional biological data

The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from all test solution concentrations appeared normal.

Validity criteria

The validity criteria specified in the OECD 201 guideline are;

1) To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 h test period. In this test, cell particle density increase (measured as a surrogate for biomass) was 90.72 over the 72 h for the control.

2) The mean coefficients of variation for control replicate sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 9.6%.

3) The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and in this test, was calculated to be 1.7%.

Based on the study results, it was concluded that the study has fulfilled validity criteria.

Validity criteria fulfilled:

yes

Conclusions:

Based on results of the read across study, the 72 h ErC50, ErC10, EyC50, EyC10, NOEC and LOEC values for the read across substance with freshwater green algae, were determined to be >19, 2.35, 3.15, 1.57, 1.5 and 3.8 mg/L, respectively

Executive summary:

A study was conducted to determine the acute toxicity study of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the read across substance were employed. Each replicate test vessel was inoculated with 0.862 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.474 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to read across substance at nominal concentrations of 0, 0.097, 2.13, 4.7, 10.3 and 23 mg/L in AAP medium for 72 h. The exposure levels of read across substance in aqueous samples of test media were monitored using a LC/MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the substance was found to be 0, 0, 1.5, 3.8, 0.66, 8.5 and 19 mg/L. The test results were expressed in terms measured concentration of the substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The ErC50 and ErC10 values (for growth rate) and EyC50 and EyC10 values (for cell particle density) were calculated to be >19, 2.35 and 3.15, 1.57 mg/L respectively. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values were determined to be 1.5 mg/L and 3.8 mg/L, respectively, for both growth rate and cell particle densities. The study results were found to have fulfilled the all the Guideline required validity criteria. Under the study conditions, 72 h ErC50, ErC10, EyC50, EyC10, NOEC and LOEC values for the read across substance with freshwater green algae, were determined to be >19, 2.35, 3.15, 1.57, 1.5 and 3.8 mg/L, respectively (Scymaris, 2018). Based on the results of the read across study, a similar EC50 and NOEC values can be expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE'.

Description of key information

Based on results of the read across study, the 72 h ErC50, ErC10, EyC50, EyC10, NOEC and LOEC values for the test substance with freshwater green algae, are considered to be >19, 2.35, 3.15, 1.57, 1.5 and 3.8 mg/L, respectively

Key value for chemical safety assessment

EC50 for freshwater algae:

19 mg/L

EC10 or NOEC for freshwater algae:

2.35 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the read across substance were employed. Each replicate test vessel was inoculated with 0.862 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.474 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to read across substance at nominal concentrations of 0, 0.097, 2.13, 4.7, 10.3 and 23 mg/L in AAP medium for 72 h. The exposure levels of read across substance in aqueous samples of test media were monitored using a LC/MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the substance was found to be 0, 0, 1.5, 3.8, 0.66, 8.5 and 19 mg/L. The test results were expressed in terms measured concentration of the substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture.The ErC50 and ErC10 values (for growth rate) and EyC50 and EyC10 values (for cell particle density) were calculated to be >19, 2.35 and 3.15, 1.57 mg/L respectively. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values were determined to be 1.5 mg/L and 3.8 mg/L, respectively, for both growth rate and cell particle densities. The study results were found to have fulfilled the all the Guideline required validity criteria. Under the study conditions, 72 h ErC50, ErC10, EyC50, EyC10, NOEC and LOEC values for the read across substance with freshwater green algae, were determined to be >19, 2.35, 3.15, 1.57, 1.5 and 3.8 mg/L, respectively(Scymaris, 2018).Based on the results of the read across study, a similar EC50 and NOEC values can be expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE'.