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Ecotoxicological information

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:
2017-06-02 - 2017-08-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study; EU method; GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to guideline
Guideline:
other: OECD No 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters
Molecular formula:
unspecified
IUPAC Name:
Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters
Test material form:
liquid
Specific details on test material used for the study:
Name: Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters
Batch No.: RP GW 915 MU
Aggregate State at Room Temperature: Liquid
Colour: Light amber

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
The samples were diluted by a factor of two with acetonitrile.
One additional sample of the control and of the dilution solvent was taken at test start and test end without any sample treatment.

Test solutions

Vehicle:
no
Details on test solutions:
Test Concentrations: 300, 95, 30, 9.5 and 3.0 mg test item/L and a control.
Dosage of Test Item:
All test concentration were prepared separately by dissolving:
3 mg/L: 15.1 mg in 5033 mL test water
9.5 mg/L: 9.6 mg in 1010.5 mL test water
30 mg/L: 15.0 mg in 500 mL test water
95 mg/L: 47.4 mg in 498.9 mL test water
300 mg/L: 150.7 mg in 502.3 mL test water
by intense stirring for 23 hours and 10 to 15 minute. The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species:Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
21.6 to 21.9 °C
pH:
8.0 to 8.2 at test start and
7.8 to 10.1 at test end
Nominal and measured concentrations:
Test Concentrations: 300, 95, 30, 9.5 and 3.0 mg test item/L and a control.
Details on test conditions:
Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. Water temperature was: 21.6 to 21.9 °C
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH was: 8.0 to 8.2 at test start and 7.8 to 10.1 at test end
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5353 lux (range: 4890 to 5670 lux)
Reference substance (positive control):
yes
Remarks:
For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
65.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
23.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
9.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
256 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
41.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
95 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
33.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
77 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The 72-hour EyC50 was calculated to be 65.4 mg test item/L and the ErC50 256 mg test item/L. The 72-hour EyC10 was calculated to be 23.2 mg test item/L and the ErC10 41.1 mg test item/L. The 72-hour NOEyC was determined to be 9.5 mg test item/L and the associated 72-hour LOEyC of 30 mg test item/L. The 72-hour NOErC was determined to be 30 mg test item/L and the associated 72-hour LOErC is 95 mg test item/L.
Results with reference substance (positive control):
Parameter Yield
(0-72 h) [mg test item/L]
72-hour EC50 0.549
95 % conf. interval 0.490 – 0.615

72-hour EC20 0.351
95 % conf. interval 0.301 – 0.412

72-hour EC10 0.278
95 % conf. interval 0.226 – 0.341

72-hour NOEC 0.21
72-hour LOEC 0.45

Parameter Growth rate
(0-72 h) [mg test item/L]
72-hour EC50 1.12
95 % conf. interval 1.06 – 1.17

72-hour EC20 0.694
95 % conf. interval 0.646 – 0.747

72-hour EC10 0.541
95 % conf. interval 0.491 – 0.596

72-hour NOEC 0.21
72-hour LOEC 0.45

Parameter Biomass
(0-72 h) [mg test item/L]
72-hour EC50 0.567
95 % conf. interval 0.503 – 0.638

72-hour EC20 0.347
95 % conf. interval 0.295 – 0.411

72-hour EC10 0.269
95 % conf. interval 0.217 – 0.334

72-hour NOEC 0.21
72-hour LOEC 0.45
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Cell Density / Increase in Control Cultures: 163.6-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 34.3 %; Coefficient of Variation of Average Growth between Control Replicates: 1.0 %
Conclusions:
The influence of Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 65.4 mg test item/L, and the 72-hour ErC50 value was calculated to be 256 mg test item/L. The 72-hour NOEyC was determined to be 9.5 mg test item/L and the associated 72-hour LOEyC was 30 mg test item/L. The 72-hour NOErC was determined to be 30 mg test item/L and the associated 72-hour LOErC was 95 mg test item/L.
The quantification of the test item Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters in the test samples was performed using liquid chromatography with MS/MS detection. At the start of the test 2% of the nominal test concentrations were found (of the nominal concentration of 300 mg test item/L). After 72 hours test duration, 9% of the nominal value was determined (of the nominal concentration of 300 mg test item/L). During the test the algae were exposed to a mean of 5% of nominal. These recoveries display the poor solubility of the test item in presence of 100% of the test item within the test system. All reported results refer to nominal concentrations.
Executive summary:

Title: Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters: Toxicity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test

Purpose: The purpose of this test was to determine the inhibitory effect of the test item Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters on the growth of the freshwater green algae Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines/Recommendations:

OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011

Commission Regulation (EC) No 761/2009, Annex, Part C, C.3.: "Freshwater Algae and Cyanobacteria, Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

 

Material and Methods

Test Item: Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters; Batch No.: RP GW 915 MU

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

Endpoints: Yield and growth rate of the algae

Test Concentrations: 300, 95, 30, 9.5 and 3.0 mg test item/L and a control.

Test Conditions: Water temperature: 21.6 to 21.9 °C; pH values at test start 8.0 to 8.2, at the end of the test 7.8 to 10.1; continuous illumination; mean light intensity: 5353 lux (4890 to 5670 lux).

The quantification of the test item Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters in the test samples was performed using liquid chromatography with MS/MS detection. At the start of the test 2% of the nominal test concentrations were found (of the nominal concentration of 300 mg test item/L). After 72 hours test duration, 9% of the nominal value was determined (of the nominal concentration of 300 mg test item/L). During the test the algae were exposed to a mean of 5% of nominal. These recoveries display the poor solubility of the test item in presence of 100% of the test item within the test system.All reported results refer to nominal concentrations.

Conclusion:

The influence of Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 65.4 mg test item/L, and the 72-hour ErC50 value was calculated to be 256 mg test item/L. The 72-hour NOEyC was determined to be 9.5 mg test item/L and the associated 72-hour LOEyC was 30 mg test item/L. The 72-hour NOErC was determined to be 30 mg test item/L and the associated 72-hour LOErC was 95 mg test item/L.

This study is classified acceptable and satisfies the guideline requirements for growth inhibition algae studies.