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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 15 Aug 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted in 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Gewerbeaufsichtsamt Hildesheim
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples were taken at the beginning and at the end of the test. Separate replicates for the test item analysis after 0 h were prepared. At the end of the test samples were taken directly from the test replicates.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 100 mg/L test solution was prepared by adding of test substance to dilution water and shaking with 20 rpm for 24 h at room temperature. After a separation phase of 2 h the final test solution was taken from the homogeneous liquid phase.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: SAG 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE/m2*s for 24h per day.

ACCLIMATION
- Acclimation period: 4 day old
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not stated
Test temperature:
21 - 24 °C
pH:
7.87 - 7.98 at test start
7.87 - 9.12 at end of test
Nominal and measured concentrations:
Nominal: 100 mg/L loading rate as filtered test solution
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Size, fill volume: 250 mL, 100 mL
- Initial cells density: 2000 - 5000 cells/mL
- Control end cells density: 609568 cells/mL (mean)
- No. of vessels per concentration: 6
- No. of vessels per control: 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: yes
- Intervals of water quality measurement: The pH-value at the beginning of the test was measured out of one additional replicate of each loading level and control. At the end it was measured from one pool per loading level of all replicates.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuously light
- Light intensity and quality: 60 - 120 µE/m2*s

EFFECT PARAMETERS MEASURED: Algal cell density was recorded after 24, 48 and 72 h exposure time by measuring of Chlorophyll-a-fluorescence. Additionally, microscopic evaluation of the cells was carried out at the start and the beginning of the test.
- Chlorophyll measurement: fluorescence, excitation at 436 nm, emission at 685 nm.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: low water solubility of the test substance
- Range finding study: performed
- Test concentrations: 1.25, 12.5 and 125 mg/L
- Results used to determine the conditions for the definitive study: No inhibition of growth rate was observed. Thus, a limit test at 100 mg/L was conducted as final test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 2.99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 2.99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: WAF
Basis for effect:
growth rate
Results with reference substance (positive control):
The EL50 value (growth rate) of the reference substance was determined to be 0.74 mg/L.

Table 1: Measured concentrations of the test susbstance

Nominal test concentration [mg/L]

Measured concentration [mg/L]

Geometric mean measured concentration [mg/L]

0h

72h

 

Control

< LOQ

< LOQ

 

100

8.85*

1.01*

2.99

*: Mean value of three replicates

Table 2: Results of the range-finding test

Nominal loading level [mg/L]

Specific growth rate inhibition [%]

Yield inhibition [%]

125

-5

-27

12.5

-2

-11

1.25

-8

-55

Table 3: Yield and growth rates and their inhibition after 72h exposure time

Nominal test item loading rate [mg/L]

Replicate No.

Yield

Yield inhibition [%]

Growth rate [d-1]

Rate related inhibition [%]

100

1

54077

10.69

1.572

2.13

2

562114

7.04

1.585

1.31

3

524121

13.32

1.562

2.74

4

605479

-0.13*

1.610

-0.22*

5

577645

4.47

1.594

0.74

6

584256

3.38

1.598

0.51

Mean

565615

6.46

1.587

1.20

Control

1

539045

 

1.571

 

2

618394

1.617

3

694244

1.655

4

463182

1.521

5

628114

1.622

6

685161

1.651

Mean

604690

1.606

*: negative inhibition = increase in yield/growth

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Jan - 23 Jan 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study with acceptable restrictions: Initial measured concentration was used to evaluate NOEC and EC50 in spite that the concentration after 72 hours was notably lower than at inition and not within 20 % of nominal concentration. Analytical purity is only 77.2%.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Nominal concentration was used to evaluate NOEC and EC50 in spite that the concentration after 72 hours was not within 20 % of nominal concentration.
GLP compliance:
yes
Remarks:
according to the Japanese GLP Standard
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples were taken directly from each vessel.
- Sample storage conditions before analysis: no storage before analysis
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substance was measured and mixed with vehicle. Purified water was added bit by bit to prepare the stock solution (1000 mg/L). Stock solution was diluted with test media.
- Controls: blank control and vehicle control
- Chemical name of vehicle: HCO-40
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 10000 mg/L in a stock solution, 1 mg/L in final concentration
- Evidence of undissolved material: Test water was clear and colourless at the study initiation.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: algae
- Strain: ATCC22662
- Source: American Type Culture Collection
- Age of inoculum: ≤ 3 days
- Method of cultivation: on a shaker

ACCLIMATION
- Acclimation period: 16 Jan - 20 Jan 2004
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.8 - 23.2; (23 ± 2 °C)
pH:
7.9 - 10.0
Nominal and measured concentrations:
Nominal concentration: 0.100 mg/L
Measured concentration: 0.0845 mg/L at the study initiation, 0.0019 mg/L at the end of the study
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer-flask
- Type: closed with air-permeable cap
- Material: glass; Size: 300 mL; Fill volume: 100 mL
- Initial cells density: 10000 cells/mL
- Control end cells density: 3056200 ± 155000 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per vehicle control: 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature and ph were measured at 24, 48 and 72 hours after test begin.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no adjustment
- Photoperiod: continuously
- Light intensity and quality: 4000 lux

EFFECT PARAMETERS MEASURED: Algal growth was recorded after 72h test duration.
- Determination of cell concentrations: counting chamber; electronic particle counter

TEST CONCENTRATIONS
- Range finding study: performed
- Test concentrations: 0.1 mg/L
- Results used to determine the conditions for the definitive study: Growth rate was 92% compared with control group and 94 % with vehicle control group. The measured concentration was 0.001 mg/L after 72 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (analytical grade)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.085 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.085 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.085 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.085 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No abnormality was observed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.432 ± 0.074 mg/L
Reported statistics and error estimates:
Student's t-test for NOEC

Table 1: Measured concentration of the test substance

Nominal Conc.

[mg/L]

Measured Conc. [mg/L], (Percent of nominal)

0 h

72 h

Control

<0.1

<0.1

Solvent control

<0.1

<0.1

0.100

0.0845

(85)

0.0019

(2)

Table 2: Growth inhibition [%]

Nominal Concentration

[Measured conc. at 0 h]

[mg/L]

Biomass

Growth rate (24-48 h)

Growth rate (24-72 h)

Inhibition [%] *

Inhibition [%] *

Inhibition [%] *

Control

-

-

-

Solvent control

-

-

-

0.100

[0.0845]

- 6.5

2.4

- 0.4

*: Values are the growth inhibition [%] relative to the solvent control.

Description of key information

No toxic effects up to the limit of water solubility (< 0.05 mg/L in aqua dest) for algae (OECD 201) based on read across.

Key value for chemical safety assessment

Additional information

No information on the toxicity of Diisodecyl azelate (CAS 28472-97-1) to algae is available. Thus in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 Grouping of substances, read-across to the structurally related substances Bis(2-ethylhexyl) azelate (CAS 103-24-2) and Bis(2-octyldodecyl) azelate (CAS 897626-46-9) was conducted. The test substance is described as an unknown, variable composition, complex reaction product or biological material (UVCB) under REACH. Further justification is given within the overall summary and within the analogue justification (IUCLID section 13).

The static test with the source substance Bis(2 -ethylhexyl) azelate (CAS 103-24-2) was conducted by a Japanese authority according to GLP and OECD guideline 201 using Pseudokirchneriella subcapitata as test organism (Mitsubishi Chemical Safety Institute Ltd., 2004). A technical product of the substance (77.2% purity) was tested at a nominal test concentration of 0.1 mg/L using HCO-40 as vehicle. The initial test concentration was measured to be 0.0845 mg/L. The concentration after 72 hours was notably lower and not within 20 % of nominal concentration. The EC50 (72 h) for growth rate is determined to be > 0.0845 mg/L and the NOEC (72 h) for growth rate is determined to be ≥ 0.0845 mg/L on the basis of the initial test concentration, which can be considered as highest achievable test substance concentration using a vehicle under test conditions.

The study with the second source substance, Bis(2 -octyldodecyl) azelate (CAS 897626-46-9), was conducted according to GLP and OECD guideline 201 using Desmodesmus subspicatus as test organism (Dr. U. Noack-Laboratorien, 2008). The test solution was taken from the liquid phase of a 100 mg/L loading rate stock solution after a 2 h separation phase. The test concentration was measured using LC-MS; however, the analysis showed that the substance is only poorly soluble in water and the concentration not stable throughout the test period. Thus, the nominal concentration was used for determination of an effect value. No significant effect on growth rate was observed after 72 hours exposure time. Hence, the EC50 (72 h) and EC10 (72 h) are determined to be > 100 mg/L loading rate.

Due to structural and property similarities to the analogue substances Bis(2-octyldodecyl) azelate (CAS 897626-46-9) and Bis(2 -ethylhexyl) azelate (CAS 103-24-2), these results are also applicable to read across to Diisodecyl azelate (CAS 28472-97-1). Therefore, Diisodecyl azelate is expected to exhibit no adverse effects towards algae in the range of water solubility.