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Ecotoxicological information

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:
2017-05-22 to 2017-05-26
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:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
March 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20160505 / 116727
- Expiration date of the lot/batch: May 2017
- Purity test date: 30.01.2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.25, 12.5, 25.0, 50.0 and 100 mg/L
- Sampling method: For the analysis of the test item concentrations, samples of each test concentration and control were taken from the test solutions at the start and at the end of the study. Three parallel samples were analysed from each test concentration and control at the start and at the end of the test. The samples were prepared immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. The test solution was freshly prepared at the beginning of the experiment, in the testing laboratory.
A stock solution of 100 mg/L (nominal concentration) was prepared by dissolving 0.08 g of the test item in 800 mL OECD medium. The pH value (5.57) of this stock solution was adjusted to the pH value of the OECD medium (7.67). The adjusted pH level of the stock solution was 7.93. The further test solutions were prepared by appropriate dilution of this stock solution. The test solutions were prepared just before introduction of algae (start of the experiment).
- Controls: Yes. Untreated control ran parallel in the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapita and Selenastrum capricornutum) (Printz-Starr).
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days at this test.)

ACCLIMATION
- Acclimation period: Four days

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.8 – 23.4 °C (measured in the flask)
22.0 – 23.7 °C (measured within the climate chamber)
pH:
7.55 – 8.94
Nominal and measured concentrations:
Nominal: 6.25, 12.5, 25.0, 50.0 and 100 mg/L
Measured: The measured concentration of the test item in the test solutions was in the range of 97 – 100 % at the start and 98 – 101 % at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the biological results was based on the nominal concentration values.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume with 100 mL test medium.
- Initial cells density: 10^4 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes. The pH value (5.57) of this stock solution was adjusted to the pH value of the OECD medium (7.67). The adjusted pH level of the stock solution was 7.93.
- Photoperiod: Continuously illumination
- Light intensity and quality: The average light intensity measured at the position occupied by algal culture flasks during the test was 8012 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Morphological Changes of Algal Cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.


PRE-EXPERIMENTS:
In order to select appropriate test concentrations for use in the definitive test, two non-GLP preliminary range-finding tests were conducted to determine the approximate toxicity of the test item.
In the first preliminary test a stock solution of 100 mg/L (nominal concentration) was prepared by dissolving the test item in OECD medium. Untreated control ran parallel in the test.
Algal cells were exposed to the concentration of the test item plus control, for 72 hours. The test was performed with three replicates in treatment group and three replicates in the control. The test item itself caused a change to the pH of the test medium. The pH of the stock solution (100 mg/L) was acid (pH: 5.64) in the first preliminary test this could cause the strong inhibitory effect therefore the second preliminary test was divided into two parts.
For preparation of test solutions the first stock solution was prepared by dissolving an amount of 0.04 g test item in 400 mL OECD medium to obtain the 100 mg/L nominal concentration. The pH value of this stock solution was adjusted to the pH value of the OECD medium (7.52). The further test solutions were prepared by appropriate dilution of this stock solution. For preparation of test solutions the second stock solution was prepared by dissolving an amount of 0.04 g test item in 400 mL OECD medium to obtain the 100 mg/L nominal concentration. The pH value of this stock solution was not adjusted. The further test solutions were prepared by appropriate dilution of this stock solution. Algal cells were exposed to each concentration of the test item plus control, for 72 hours. The test was performed with two replicates in treatment groups and three replicates in the control. The control group was common for the pH adjusted and the other test solutions.
- Results used to determine the conditions for the definitive study: Based on the results of the non-GLP Preliminary Range-Finding Tests five test concentrations in a geometric series (with a spacing factor of 2.0) were used in the main test. Untreated control ran parallel in the test. The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0, 100 mg/L (loading rate).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: >100 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
72.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% conf. limits: 57.41 - 91.45 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: >100 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% conf. limits: 156.36 - 627.25 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Results with reference substance (positive control):
- Results with reference substance valid? Yes
The 72h ErC50: 0.99 mg/L, (95 % confidence limits: 0.68 – 1.49 mg/L)
The 72h EyC50: 0.59 mg/L, (95 % confidence limits: 0.47 – 0.75 mg/L)

Table 1:Growth Rates (µ) and percentage inhibition of µ during the test period

Nominal concentration

[mg/L]

Growth Rates (µ) and % Inhibition of µ

0-24 h

0-48 h

0-72 h

µ

%

µ

%

µ

%

Untreated Control

0.0478

-

0.0552

-

0.0563

-

6.25

0.0458

4.2

0.0564

-2.1

0.0565

-0.3

12.50

0.0498

-4.2

0.0554

-0.4

0.0568

-0.9

25.0

0.0345

27.8

0.0498

9.8

0.0566

-0.6

50.0

0.0458

4.2

0.0464

15.9

0.0553

1.7

100.0

0.0401

16.0

0.0492

10.9

0.0539

4.3

 

Table 2:Yield (y) and percentage inhibition of y during the test period

Nominal concentration

[mg/L]

Yield y and % Inhibition of y

0-72 h

y

%

Untreated control

56.50

-

6.25

57.33

-1.5

12.5

58.67

-3.8

25.0

58.00

-2.7

50.0

52.67

6.8

100.0

47.33

16.2

 

Validity Criteria of the Study
The experiment was considered to be valid because:
- cell density in the control cultures increased by a factor of more than 16 (by a factor of 57.50) within 72 hours.
- The mean coefficient of variation for section-by-section specific growth rates(days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %.
CV for section-by-section growth rate day 0-1: 10.24 %
CV for section-by-section growth rate day 1-2: 8.90 %
CV for section-by-section growth rate day 2-3: 6.20 %
- The mean coefficient of variation for section-by-section specific growth rates: 8.45 %.  the coefficient of variation of average specific growthrates during the whole test period in replicate control cultures did not exceed 7 % in the test.  
CV for average specific growth rate day 0-3: 1.09 %

Table 3: Summarised measured concentrations of the test itemin formulation samples

Sampling date

Nominal concentration (mg test item/L)

Mean of the measured concentration (mg test item/L)

Measured concentration in percentage of the nominal

Relative Standard Deviation % (n=3)

22 May 2017

control

not detected

-

-

6.25

6.24

100

1

12.5

12.31

99

0

25

24.31

97

0

50

48.92

98

0

100

97.51

98

1

25 May 2017

control

not detected

-

-

6.25

6.10

98

1

12.5

12.45

100

1

25

25.02

100

0

50

50.35

101

0

100

101.00

101

1

 

 

Conclusions:
In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item, 3-Tert Butyladipic Acid, had significant toxic effects on the growth of Raphidocelis subcapitata at the concentration of 100 mg/L. An EC10 and EC50 based on growth rate and yield of >100 mg/L was determined, respectively. The NOEC was 50 mg/L based on growth rate and yield.

Description of key information

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item, 3-Tert Butyladipic Acid, had significant toxic effects on the growth of Raphidocelis subcapitata at the concentration of 100 mg/L. An EC10 and EC50 based on growth rate and yield of >100 mg/L was determined, respectively. The NOEC was 50 mg/L based on growth rate and yield.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
50 mg/L

Additional information

An Growth Inhibition Test according to OECD 201 was conducted to determine the effect of the test item, 3 -Tert Butyladipic Acid on the growth of the unicellular green algae species Raphidocelis subcapitata(formerly known as Pseudokirchneriella subcapitata). Exponentially growing cultures of Raphidocelis subcapitata were exposed to test item concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg/L. The algal growth in relation to the untreated control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. An EC10 and EC50 based on growth rate and yield of >100 mg/L (nominal) was determined, respectively. The NOEC was 50 mg/L (nominal) based on growth rate and yield. The measured concentration of the test item in the test solutions was in the range of 97 – 100 % at the start and 98 – 101 % at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the biological results was based on the nominal concentration values.