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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:
2018-05-17 to 2018-08-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study; EU method; GLP
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
other: OECD No 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: TEMPIC
Synonym: THIOCURE TEMPIC
Chemical Name: Tris[2-(3-mercaptopropionyloxy)ethyl]isocyanurat
CAS No.: 36196-44-8
Batch No.: 0061488
Purity: 95.9 % according to certificate of analysis
Manufacturing Date: December 21, 2017
Certificate of Analysis Date: December 20, 2017
Aggregate State at Room Temperature: Liquid
Colour: Light yellow
Expiry Date: December 20, 2018
Storage Conditions at Test Facility: At 4 +/- 4 °C, in the dark
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 after 24 hours and at the end of the test (after the 72 hours test period) by pooling aliquots of the contents of the test beakers of each treatment.
All samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.
Vehicle:
no
Details on test solutions:
A defined amount of the test item was added directly to the test water for each test concentration and was carefully stirred for 24 hours in the dark to dissolve as much of the test item as possible. The highest test item concentration of 100 mg test item/L was prepared by mixing 101.5 mg test item into 1015 mL test water, for the test item concentration of 32 mg test item/L, 32.1 mg test item were mixed into 1003 mL test water, for the concentration of 10.0 mg test item/L, 10.1 mg were mixed into 1010 mL test water. The concentration of 3.20 mg test item/L was prepared by mixing 6.5 mg into 2031 mL test water and for 1.0 mg test item/L, 5.0 mg were mixed into 5000 mL test water. After cessation of mixing and a following period (1 hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration. The test media were prepared just before introduction of the algae (= start of the test).
Test Concentrations:
Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2, 1.0 mg test item/L and a control were tested corresponding to following geometric mean measured concentrations of the test item: 21.9, 9.84, 5.51, 1.25 and 0.319 mg test item/L and a control.
Control: In the control, test water was used without addition of the test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
22 to 23°C
pH:
7.8 - 8.0 at test start and 7.9 to 9.1 at test end
Nominal and measured concentrations:
Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2, 1.0 mg test item/L and a control were tested corresponding to following geometric mean measured concentrations of the test item: 21.9, 9.84, 5.51, 1.25 and 0.319 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: 22.1 to 23.0 °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: 7.8 to 8.0 at test start and 7.9 to 9.1 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: 6203 lux (range: 5940 to 6470 lux)
Reference substance (positive control):
yes
Remarks:
reference item potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
5.51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.319 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Growth Inhibition: The 72-hour EyC50 was calculated to be 3.51 mg test item/L and the ErC50 13.0 mg test item/L. The 72-hour EyC10 was calculated to be 1.14 mg test item/L and the ErC10 3.00 mg test item/L. The 72-hour NOEyC was determined to be 0.319 mg test item/L and the associated 72-hour LOEyC of 1.25 mg test item/L. The 72-hour NOErC was determined to be 1.25 mg test item/L and the associated 72-hour LOErC is 5.51 mg test item/L.
Microscopic Examination: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal loading rate of 100 mg test item/L (geometric mean measured of 21.9 mg test item/L) and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
Results with reference substance (positive control):
Parameter (0-72 h) Yield [mg test item/L] Growth rate [mg test item/L] Biomass [mg test item/L]
72-hour EC50 0.402 0.963 0.414
95 % conf. interval 0.373 - 0.434 0.927 - 1.001 0.385 - 0.446

72-hour EC20 0.225 0.513 0.224
95 % conf. interval 0.197 - 0.250 0.482 - 0.543 0.198 - 0.248

72-hour EC10 0.166 0.369 0.163
95 % conf. interval 0.139 - 0.191 0.339 - 0.398 0.137 - 0.186

72-hour NOEC 0.094 0.094 0.094
72-hour LOEC 0.21 0.21 0.21
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by using the 3 parametric concentration distribution function (CDF).
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 (yield) and Bonferroni-Welch t-test (growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.
Validity criteria fulfilled:
yes
Remarks:
Cell Density Increase: 159.3-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates: 15.1 %; Coefficient of Variation of Average Growth: 1.0 %
Conclusions:
The influence of TEMPIC 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 3.51 mg test item/L, and the 72-hour ErC50 value was calculated to be 13.0 mg test item/L. The 72-hour NOEyC was determined to be 0.319 mg test item/L and the associated 72-hour LOEyC was 1.25 mg test item/L. The 72-hour NOErC was determined to be 1.25 mg test item/L and the associated 72-hour LOErC was 5.51 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal or measured initial concentrations during the test.
Executive summary:

Title: TEMPIC: 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 TEMPIC 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

Materials and Methods

Test Item: TEMPIC; Batch No.: 0061488; purity: 95.9 %; according to certificate of analysis

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

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 for determination of cell densities by spectrophotometrical measurement.

Endpoints: Yield and growth rate of the algae

Test Concentrations: Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2, 1.0 mg test item/L and a control were tested corresponding to following geometric mean measured concentrations of the test item: 21.9, 9.84, 5.51, 1.25 and 0.319 mg test item/L and a control.

Test Conditions: Water temperature: 22.1 to 23.0 °C; pH values at test start 7.8 to 8.0, at the end of the test 7.9 to 9.1; continuous illumination; mean light intensity: 6203 lux (5940 to 6470 lux).

Results

Biological Results:

Yield:

The 72-hour EC50 was calculated to be 3.51 mg test item/L. The 72-hour NOEC was determined to be 0.319 mg test item/L and the associated 72-hour LOEC of 1.25 mg test item/L.

Growth rate:

The 72-hour EC50 was calculated to be 13.0 mg test item/L. The 72-hour NOEC was determined to be 1.25 mg test item/L and the associated 72-hour LOEC is 5.51 mg test item/L.

Analytical Results:

The quantification of the test item TEMPIC in the test samples was performed using liquid chromatography with MS/MS detection.

At the start of the test 64 % of the nominal test concentrations were found (average of all test concentrations). After 24 and 72 hours test duration, 52 % and 20 % of the nominal value was determined (average of all test concentrations).

Conclusion:

The influence of TEMPIC 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 3.51 mg test item/L, and the 72-hour ErC50 value was calculated to be 13.0 mg test item/L. The 72-hour NOEyC was determined to be 0.319 mg test item/L and the associated 72-hour LOEyC was 1.25 mg test item/L. The 72-hour NOErC was determined to be 1.25 mg test item/L and the associated 72-hour LOErC was 5.51 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal or measured initial concentrations during the test.

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

Description of key information

OECD TG 201 study conducted with TEMPIC.

Key value for chemical safety assessment

EC50 for freshwater algae:
13 mg/L
EC10 or NOEC for freshwater algae:
1.25 mg/L

Additional information

A NOEC = 1.25 mg/L could be reliable reported from this study.