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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 13, 2017 to November 16, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
High-performance liquid chromatography / mass spectrometry (HPLC/MS) method
Details on sampling:
To demonstrate that nominal exposure concentrations were being achieved, the concentrations of test substance in the test solutions were measured using the high-performance 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, solvent control and nominal exposure concentrations of 0.085, 0.19, 0.41, 0.91 and 2.0 mg/L. The test substance was ground to a white powder in a pestle and mortar. Primary stock concentrates of test substance with nominal concentrations of 10 and 20 g/L were prepared by weighing a nominal 0.1 g of test substance (actual weight: 0.09997 g) and 0.2 g of test substance (actual weight: 0.19993 g) respectively into 10 mL volumetric flasks and making up to volume with tetrahydrofuran (THF). The volumetric flasks were placed in an ultrasonic bath for approximately 1 h and the resultant stocks were both observed to be cloudy white solutions (classified as homogenous dispersions). Test solutions were prepared by the direct addition of the appropriate amount of concentrate to dilution water via a microliter syringe into a stirring solution in a volumetric flask. The solvent control was prepared in the same way using solvent only. The control consisted of culture medium only. In all cases the final solutions contained nutrients. All test solutions were observed as clear and colourless. A solvent was used in this study to assist in dosing the test compound due to its apparent low solubility in test media, the concentration of solvent used in all exposure solutions with the exception of the control was 100 µL/L. The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 8.04
Nominal and measured concentrations:
A 2.0 mg/L test concentration was selected as the highest concentration based on the toxicity demonstrated in a non-GLP range-finding study.
0, 0.085, 0.19, 0.41, 0.91 and 2.0 mg/L (Nominal)
0, 0.041, 0.060, 0.14, 0.20, 0.34 mg/L (Mean measured)
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 at 160 rpm.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 0.34 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.:
> 0.34 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. 0.14 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. 0.2 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 0.06 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 56-96% of nominal and at the end were 0-20% of nominal. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value 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 two‑sample t test to identify significant differences (p<0.05) between the control and solvent control. There was no significant effect (p=0.9295) between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). ECXwas calculated using the linear interpolation (ICPIN) method. 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

0.14

-

LOEC

0.20

-

ErC50

>0.34

N/A

ErC20

>0.34

N/A

ErC10

0.18

0.15-0.24

Yield

This response was 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 two-sample t test to identify significant differences (p<0.05) from the control and solvent control. There was no significant effect (p=0.9651) between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). ECX was calculated using the linear interpolation (ICPIN) method. The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method. 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

0.14

-

LOEC

0.20

-

EyC50

>0.34

N/A

EyC20

0.16

0.13-0.18

EyC10

0.15

N/A

Additional biological data

The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the solvent control and all test 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 66.4 and 66.2 over the 72 h for the control and solvent control respectively.

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 15%.

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 2.1% and 3.2% for control and solvent control respectively.

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

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.34, >0.34, 0.14 and 0.20 mg/L, respectively.
Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' 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 test substance were employed. Each replicate test vessel was inoculated with 0.440 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 0.515 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.085, 0.19, 0.41, 0.91 and 2.0 mg/L in AAP medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC/MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.041, 0.060, 0.14, 0.20 and 0.34 mg/L. The test results were expressed in terms measured concentration of test 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) were found to be >0.34 and 0.18 mg/L and EyC50 and EyC10 values (for cell particle density) were found to be >0.34 and 0.15 mg/L respectively. The NOEC and the LOEC values were found to be 0.14 mg/L and 0.20 mg/L, respectively, for both growth rate and cell particle densities. The study results were considered to have fulfilled the all the validity criteria. Under the study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.34, 0.18, >0.34, 0.15, 0.14 and 0.20 mg/L, respectively (Scymaris, 2017).

Description of key information

Based on the results of the ready across study, similar absence of significant toxicity is expected up to thehighest soluble concentration of the test substance. As a conservative approach, the 72 h ErC50, ErC10, and NOEC values of the test substance for toxicity to freshwater algae is considered to be >0.34, 0.18 and 0.14 mg/L respectively (Scymaris, 2017).

Key value for chemical safety assessment

EC50 for freshwater algae:
0.34 mg/L
EC10 or NOEC for freshwater algae:
0.18 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the read across substance, ‘phosphoric acid, hexadecyl esters, potassium salts with cetyl alcohol and isostearyl isostearate’ 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 0.440 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 0.515 × 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, 0.085, 0.19, 0.41, 0.91 and 2.0 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 HPLC/MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the read across substances were found to be 0, 0.041, 0.060, 0.14, 0.20 and 0.34 mg/L. The test results were expressed in terms measured concentration of read across 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) were found to be >0.34 and 0.18 mg/L and EyC50 and EyC10 values (for cell particle density) were found to be >0.34 and 0.15 mg/L respectively. The NOEC and the LOEC values were found to be 0.14 mg/L and 0.20 mg/L, respectively, for both growth rate and cell particle densities. The study results were considered to have fulfilled the all the validity criteria. Under the study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, NOEC and LOEC values for the read across substance with freshwater green algae, were determined to be >0.34, 0.18, >0.34, 0.15, 0.14 and 0.20 mg/L, respectively (Scymaris, 2017). Based on the results of the ready across study, similar absence of significant toxicity is expected up to the highest soluble concentration of the test substance.