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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:
2019-03-29 to 2019-05-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling: 4 sampling time points of at each 5 test concentrations + negative control (NC) samples, 24 samples prediluted 1:2 with acetonitrile (ACN)
- Substance specific analysis was performed at the test site of menal GmbH.
- The sampling was conducted according to the following specification:
After 0 h, 24 h, 48 h and 72 h exposure, both additional vessels of NC, A, B, C, D and E were sampled; two samples per replicate: 4 samples of 1 mL per treatment group.
For each sampled treatment, two of the analytic samples from 0 h, 24 h, 48 h and 72 h were sent to the analytical laboratory for chemical analysis using an insulated box with thermal packs.
The remaining samples were stored as retain samples in the freezer (≤ -18 °C) until finalization of the study.
- Concentrations: NC (nominal 0 mg/L), A (nominal 0.1 mg/L), B (nominal 0.3 mg/L), C (nominal 0.9 mg/L), D (nominal 2.7 mg/L) and E (nominal 8.1 mg/L)
- Sampling method: The samples were filled into 8 mL glass vials. To each vial, 1 mL acetonitrile was added and samples were stored in the freezer (≤ -18 °C).
- Sample storage conditions before analysis: Samples were stored at -20 °C ± 10 °C at menal GmbH until analysis (in medium/ACN, 50/50, v/v) at least until finalization of the phase report after which they will be destroyed or further stored. On assay day, samples were thawed at room temperatures and directly transferred into an HPLC-vial.
- Shipping: Personal handover between Hydrotox GmbH and menal GmbH
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution was prepared by adding 9.0 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 1000 mL test medium and shaking for 24 hours using an overhead shaker at 22.8 - 23.5 °C in the dark. This stock solution was used as highest test concentration in the test. Into each test vessel for the test item treatment and negative control (NC), 50 mL test preparation (test solution and algal inoculum) was transferred. The further test item concentrations were prepared by diluting the stock solution with test medium (dilution factor of 3).
- Controls: The negative control was the test medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, also known as Raphidocelis subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettingen
- Age of inoculum (at test initiation): 4-day-old pre-culture was used as inoculum in the test
- Method of cultivation: suspension culture

ACCLIMATION
- Acclimation period: Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium (composition: 496 mg/L NaNO3, 39 mg/L K2HPO4, 75 mg/L MgSO4×7H2O, 36 mg/L CaCl2×2H2O, 58 mg/L Na2CO3, 10 mg/L Na2EDTA×2H2O, 3 mg/L citric acid, 3 mg/L iron citrate, 0.1144 mg/L H3BO3, 0.0724 mg/L MnCl2× 4H2O, 0.0088 mg/L ZnSO4×7H2O, 0.0032 mg/L CuSO4×5H2O, 0.0010 mg/L Na2MoO4×2H2O, 0.0016 mg/L CoCl2×6H2O) under axenic conditions to keep it in exponential growth.
- Culturing media and conditions (same as test or not): 4 days before test, the same culturing conditions as in the test.
- Any deformed or abnormal cells observed: no abnormality observed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
22.1 - 23.3 °C
pH:
The pH was 7.9 in the control and 7.8 - 8.1 in the test item treatment
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal test item concentrations [mg/L]: 0.1, 0.3, 0.9, 2.7 and 8.1
Measured test item concentrations (geometric mean) [mg/L]: 0.05 (for measurements
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks 100 mL (sealed with stainless steal caps)
- Material, size, headspace, fill volume: test vessels are filled to 50 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no, static test
- Initial cells density: 7 x 10E4 cells/mL (nominal inoculum concentration)
- Control end cells density: 119.211 x 10E5
- No. of vessels per concentration (replicates): 3 replicates per concentration
- No. of vessels per control (replicates): 6 replicates per negative control

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: solutions to make the 10-fold concentrated test medium were dissolved in deionised water; 1-fold concentrated medium was prepared by diluting 10-fold medium with ultrapure water
- Intervals of water quality measurement: The pH was measured at the start and end of the test; vessels were kept in light incubator (21 - 24 °C, constant within ± 2 °C) and continuously illuminated by LEDs (warm white)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Mean light intensity was 84.5 µE m-2 s-1 ± 7.5%, supplied by LEDs

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density, every 24 h; The inhibition of algal growth was determined from yield and growth rate; calculation according to the OECD guideline 201
- Determination of cell concentrations: To convert the measured surrogate parameter chlorophyll fluorescence into a cell concentration (the measure for biomass), a correlation factor is determined twice a year within the quality check by measuring cell concentrations of different dilutions with the Coulter Counter and the corresponding fluorescence with the microplate fluorescence reader. Chlorophyll fluorescence values are correlated with measured cell concentrations. The slope of the curve gives the conversion factor. By means of the conversion factor, the surrogate parameter chlorophyll fluorescence is converted into a cell concentration
- Chlorophyll measurement: The algal biomass was monitored by measuring the chlorophyll fluorescence after 0 h, 24 h, 48 h and 72 h. From each test vessel, 200 µL test solution was transferred into a 96-well microplate and the fluorescence measured with the fluorescence microplate reader at an excitation wavelength of 465 nm and an emission wavelength of 670 nm. Each measurement was conducted in duplicate. If the variation coefficient was > 10%, the measurement was repeated. Test medium was measured as blank value and subtracted from the fluorescence values in the test vessels.

TEST CONCENTRATIONS
- Range finding study: Yes, a preliminary test without GLP was performed before start of this GLP-study
- Test concentrations (in range finding study): Nominal loading rates of 1, 10 and 100 mg/L test item were tested and resulted in inhibition of 26.2%, 96.6% and > 100% for growth rate and 63.4%, 99.7% and 102.7% for yield (72 h), respectively.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.51 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.:
0.51 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.:
0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.26 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.:
0.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Potential degradation product of the test item was observed during the pre-tests and also taken into account during the chemical analysis by manual integration of both, the peak of the test item as well as a second peak (presenting the degradation product) directly eluting after the test item (semi-quantitative approach).
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

As the measured test item concentrations are not within ±20% of the nominal concentrations, according to OECD 201 (2006), all results are given in relation to the analytically measured test item concentrations (geometric mean concentrations).

During the pre-tests for the studies with Pseudokirchneriella subcapitata, an unknown potential degradation product (probably an oxidation product of the test item, according to the sponsor) with a similar mass as the test item was observed as a second peak in the chromatogram diluting directly after the test item. This potential degradation product was not identified until this study was conducted. The potential degradation product could not be identified (additional equipment, resources and experience with regards to the behavior of the test item in the test solution (e.g. kinetics) would be required), the results are based on the parent molecule.

 

Table 1 summarizes inhibition of yield and Table 2 the inhibition of growth rate after 24 h, 48 h and 72 h exposure.

Table 1: Inhibition of yield

Nominal test item concentration [mg/L]

Inhibition of yield [%]

24 h

48 h

72 h

NC

-

-

-

0.1

-12.0

-25.4

-19.1

0.3

-38.8

-24.9

-3.9

0.9

-28.9

-16.2

10.7

2.7

0.2

7.9

52.9

8.1

12.6

75.1

94.6

Table 2: Inhibition of growth rate

Nominal test item concentration [mg/L]

Inhibition of growth rate [%]

24 h

48 h

72 h

NC

-

-

-

0.1

-7.5

-8.6

-4.4

0.3

-22.8

-8.5

-1.0

0.9

-17.2

-5.7

2.6

2.7

0.2

2.9

18.2

8.1

8.6

47.3

66.4

pH values at the start and the end of the test are provided in Table 3.

Table 3: pH values at the start and the end of the test

Nominal test item concentration [mg/L]

0 h

72 h

NC

7.9

7.9

0.1

8.0

7.9

0.3

8.1

7.9

0.9

8.1

7.8

2.7

8.1

7.8

8.1

8.1

7.8

Analytically measured test item concentrations after 0 h, 24 h, 48 h and 72 h exposure are shown in Table 4.

Table 4: Analytically measured test item concentrations

Nominal test item concentration [mg/L]

Sampling time [h]

Measured test item concentration [mg/L]

Recovery [%nominal]

Geometric mean test item concentration [mg/L]

NC

0

< LOD

-

-

24

< LOD

-

48

< LOD

-

72

< LOD

-

0.1

0

< LOD

-

0.05*

24

< LOD

-

48

< LOD

-

72

< LOD

-

0.3

0

< LOQ (0.06)

(20.0)

0.05*

24

< LOD

-

48

< LOD

-

72

< LOD

-

0.9

0

0.20

22.2

0.15

24

0.15

16.7

48

0.14

15.6

72

0.11

12.2

2.7

0

0.60

22.2

0.51

24

0.56

20.7

48

0.51

18.9

72

0.41

15.2

8.1

0

2.16

26.7

1.99

24

2.05

25.3

48

1.96

24.2

72

1.81

22.4

For measurements which are < LOD, the LOD was used for calculation of the geometric mean

Analytically measured concentrations of the test item and the potential degradation product (sum of both peaks) are illustrated in Table 5.

Table 5: Analytically measured concentrations of the test item and the potential degradation product (sum of both peaks)

Nominal test item concentration [mg/L]

Sampling time [h]

Measured sum of both peaks [mg/L]

Recovery [%nominal]

Geometric mean sum of both peaks [mg/L]

2.7

0

0.63

23.3

0.54

24

0.58

21.5

48

0.53

19.6

72

0.43

15.9

8.1

0

2.33

28.8

2.14

24

2.19

27.0

48

2.10

25.9

72

1.95

24.1

CRITERIA OF VALIDITY

- The biomass (cell concentration) in the control has increased by a factor of 54.7 and therefore ≥ 16 during the test period.

- The mean coefficient of variation for section-by-section growth rate (day 0 - 1, 1 - 2 and 2 - 3) in the control is 26.5% and therefore ≤ 35%.

- The coefficient of variation of average specific growth rate during the test period in the control replicates is 3.2% and therefore ≤ 7%.

- The test is valid according to OECD Test Guideline 201 (23 March 2006).

Validity criteria fulfilled:
yes
Conclusions:
In a Freshwater Alga and Cyanobacteria, Growth Inhibition Test according to OECD guideline 201, Pseudokirchneriella subcapitata was exposed to the test item for 72 h. An ErC50 (endpoint Growth rate) was determined to equal 1.26 mg/L. The evaluation of the endpoint Yield revealed an EbC50 of 0.44 mg/L.
Executive summary:

In a 72-hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata (Strain No. 61.81 SAG) were exposed to 1,3,5-Tris-(3-mercaptopropyl)isocyanurate at nominal concentrations of 0.1, 0.3, 0.9, 2.7, and 8.1 mg/L, and measured concentrations of <LOD (0.05), <LOD (0.05), 0.15, 0.51 and 1.99 mg/L under static conditions in accordance with the OECD guideline 201. 

The NOErC and ErC50values based on growth rate were 0.15 mg/L and 1.26 mg/L, respectively.

No abnormalities were noted.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for a Freshwater Alga and Cyanobacteria, Growth Inhibition Test.

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata

Test Type (Flowthrough, Static, Static Renewal): Static

 

Growth rate:

72-h ErC50 =1.26 mg/L [95 % C.I. = 0.97 – 1.63 mg/L]

72-h ErC10 = 0.33 mg/L [95 % C.I. = 0.26 – 0.41 mg/L]

72-h NOErC = 0.15 mg/L

72-h LOErC = 0.51 mg/L

 

Yield:

72-h EbC50 =0.44 mg/L [95 % C.I. = 0.21 – 0.94 mg/L]

72-h EbC10 = 0.13 mg/L [95 % C.I. = 0.07 – 0.26 mg/L]

72-h NOEbC =0.15 mg/L

72-h LOEbC = 0.51 mg/L

 

Endpoint(s) Effected: Growth rate and Yield

Description of key information

In a Freshwater Alga and Cyanobacteria, Growth Inhibition Test according to OECD guideline 201, cultures of Pseudokirchneriella subcapitata were exposed to the test item for 72 h. An ErC50 and NOErC (endpoint Growth rate) were determined to equal 1.26 mg/L and 0.15 mg/L (geometric mean of measured concentrations).

Key value for chemical safety assessment

EC50 for freshwater algae:
1.26 mg/L
EC10 or NOEC for freshwater algae:
0.15 mg/L

Additional information