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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
- Lot/batch No.: 001101TMO
Analytical monitoring:
yes
Details on sampling:
- Nominal concentrations: 0, 0.79, 1.57, 3.13, 6.25, 12.5, 25.0 and 50.0 mg/mL and dispersant control
- Sampling method: 0.2 - 21.0 mL sample taken from each test vessel at test start (before seeding with algae). At test end, procedure repeated, samples centrifuged and supernatant retained for analysis
- Sample storage conditions before analysis: No data
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution prepared at 100 mg/L concentration in test medium. Solubilizer, hydrogenated castor oil HCO-50 at 100mg/L, used to improve solubility/dispersion.
- Eluate: Test medium
- Controls: yes, OECD medium & OECD medium plus solubilizer, HCO-50 at 100mg/L
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): solubilizer, HCO-50 100mg/L
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): test solutions were lightly translucent but no precipitation observed
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum ATCC22662 (purchased from ATCC)
- Source (laboratory, culture collection): purchased from ATCC
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: AGP-150RL apparatus

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes, OECD medium
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
N/A, OECD medium used
Test temperature:
22.0 - 24.5 deg C, average 23.6 deg C
pH:
pH=7.3-7.5 at start and 8.1-8.3 at end of the test (72h) from 1 replicate in each concentration
Dissolved oxygen:
No data
Salinity:
N/A OECD medium used
Nominal and measured concentrations:
Nominal concentrations : 0, 0.79, 1.57, 3.13, 6.25, 12.5, 25.0 and 50.0 (mg/L)

Measured concentrations : At start of the test (0 hr), <0.006, 0.72, 1.48, -, 6.02, 13.6, 29.4 and 56.8 mg/L; at end of the test (72 hr), <0.006, 0.67, 1.34, 2.85, 5.67, 14.8, 25.2 and 44.6 mg/L respectively
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask with a silicon cap which allowed ventilation.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL OECD medium in a 300 mL Erlenmeyer flask
- Aeration: No data
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A static
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 1E4/mL
- Control end cells density: Control mean cell density increased 132-fold after 72 hr; Dispersant control mean cell density increased 129-fold
- No. of organisms per vessel: 1E6
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes - OECD medium
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Intervals of water quality measurement: 0 & 72h

OTHER TEST CONDITIONS
- Sterile test conditions: yes - filtered before use
- Adjustment of pH: no
- Photoperiod: continuous illumination:
- Light intensity and quality: 4,000-5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth inhibition 0, 24, 48 & 72h,
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Coulter counter
- Chlorophyll measurement: No

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A, limit test
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: Yes
- Test concentrations: No data
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: comparison of area under growth curve
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: comparison of area under growth curve
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes, For control mean cell density increased to 1.316E4 cells/mL after 72 hr; for dispersant control mean cell density increased to 1.289E4 cells/mL
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.41 mg/L
Reported statistics and error estimates:
Bartlett test for homogeneity in variances and One-way Anova (EcoTox-Statistics Ver.1.0 beta-edition R1.4) were used for EC50, LC50 and NOEC determination (p=0.05).
Method of calculating mean measured concentrations: Geometric mean.

Nominal

Measured concentration (mg/L)

Concentration (mg/L)

0 hours

72 hours

Control

<0.006

<0.006

Dispersant control

<0.006

<0.006

0.79

0.72

0.67

1.57

1.48

1.34

3.13

-

2.85

6.25

6.02

5.67

12.5

13.6

14.8

25.0

29.4

25.2 

50.0

56.8

44.6 

- Analysis failed

 

 

 

Nominal

Inhibition (%)

Concentration (mg/L)

Yield (Biomass)

Growth rate

Control

-

-

Dispersant control

4.29

-2.08

0.79

3.39

-0.20

1.57

7.37

-0.96

3.13

14.91

-0.13

6.25

17.44

2.83

12.5

14.91

5.36

25.0

17.22

3.19 

50.0

7.19

21.48 

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions in which this study was conducted the : the growth inhibition(comparison of area under growth curve) EC50 (0-72 h) > 50.0 mg/L NOEC (0-72 h) 50.0 mg/L. The growth inhibition (comparison of growth rates) EC50 (24-72h) > 50.0 mg/L; NOEC (24-72h) 25.0 mg/L

Executive summary:

Algal growth inhibition has been investigated in a 72 hour test according to OECD test methods. Dissolution/dispersion agents were used to increase concentration of the substance in the test medium. Dissolution/dispersion agents were used to increase concentration of the substance in the test medium.The EC50 values for inhibition of specific growth rate (ErC50) and yield (EyC50) being >50.0 mg/L. The NOEC for inhibition of specific growth rate (ErC0) was 25.0 mg/L and yield (EyC0) was 50.0 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1994-02-01 to 1994-02-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions. For read-across justification see Section 13.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
As the test substance was poorly soluble in water, a 1 g/L mixture with deionised water was prepared. This mixture was stirred for 18 hours and filtrated afterwards. The filtrate was analysed and showed an initial content of 3.9 mg/L DOC and was used as stock solution.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: Scenedesmus subspicatus (CHODAT (86.81 SAG))
- Source (laboratory, culture collection): Institut für Wasser-, Boden- und Lufthygiene, Berlin; subsequently in-house breeding
- Method of cultivation: in sterile-aerated Erlenmeyer flasks on light benches


ACCLIMATION
- Acclimation period: A preculture is withdrawn from a stock culture by transfer 3 days prior to the beginning of the test from which preculture the test cultures are inoculated at a cell density of approximately 20 000 cells/mL.
- Culturing media and conditions (same as test or not): not mentioned
- Any deformed or abnormal cells observed: not mentioned
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
Growth/test medium chemistry as described in guideline 92/69/EEC (C.3.), no further details mentioned
Test temperature:
24 ± 2 °C
pH:
8.0 - 8.9 (start of test)
8.7 - 9.3 (end of test)
Dissolved oxygen:
not mentioned
Salinity:
according to guideline
Nominal and measured concentrations:
Nominal: 0.24 / 0.39 / 0.67 / 1.2 / 2.0 / 3.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile-aerated Erlenmeyes flasks
- Initial cells density: 2x10E4 cells/mL
- Control end cells density: 88x10E4 cells/mL
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates): not applicable


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
no details mentioned


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not adjusted
- Photoperiod: not specified
- Light intensity and quality: approx. 8000 lux, white


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell numbers for exposure times of 24, 48, 72 h
- Determination of cell concentrations: The cell numbers were determined photometrically at a wavelength of 685 nm
- Chlorophyll measurement: no
- Other: none


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not mentioned
- Range finding study: not mentioned
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 3.5 mg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no observations made
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no


Results with reference substance (positive control):
no reference substance tested
Reported statistics and error estimates:
no statistical evaluation performed

Table #1: Cell density data:

 concentration

[mg/L]

 cell number (x 10E4 cells/mL) after        
  0 h   24 h  48 h  72 h
 control  2  13 28  88 
0.24   2  13 34 100 
 0.39  2  13 33  98 
 0.67  2  13 32  97 
 1.2  2  13 35  109
 2.0  2  13 34  104
 3.5  2  14 38  117

                                                                                 

Table #2: Growth curves:

concentration [mg/L]   biomass (area under the growth curve) inhibition of biomass [%]   growth rate µ (0 - 72 h)  inhibition of growth rate [%]
 control 80   -  1.261  -
 0.24  92  -15  1.304  -3.4
 0.39  90  -12.5  1.297  -2.9
 0.67  88.5  -10.6  1.294  -2.6
 1.2  97.5  -21.9  1.333  -5.7
 2.0  94  -17.5  1.317  -4.4
 3.5  105.5  -31.9  1.356  -7.5

negative values indicating an activation of growth

Validity criteria fulfilled:
yes
Conclusions:
Not toxic under test conditions in the range of water solubility.
Executive summary:

The inhibitory effect of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters on the growth of the green alga Scenedesmus subspicatus, strain CHODAT (86.81 SAG) had been tested in accordance with OECD Guideline for Testing of Chemicals No.201 and EEC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC.

The test algae were exposed to 6 concentrations of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters, ranging from 0.24 to 3.5 mg/L. As the test substance was poorly soluble in water, a 1 g/L mixture with deionised water was prepared. This mixture was stirred for 18 hours and filtrated afterwards. The filtrate was analysed and showed an initial content of 3.9 mg/L DOC and was used as stock solution. The cultures were incubated on sterile ventilated Erlenmeyer flasks on light tables at 24±2 °C. Growth was monitored by determining the cell number photometrically at a wave length of 685 nm after 24, 48 and 72 hours. No inhibitory effects on the growth of the green alga were observed.

On the basis of cell growth, a mean effective concentration was calculated to be EbC50 (72 h) > 3.5 mg/L.

On the basis of the growth rate, a mean effective concentration was calculated to be ErC50 (0 -72 h) > 3.5 mg/L.

The NOEC value was >= 3.5 mg/L (on basis of cell growth).

Description of key information

Toxicity to aquatic algae: EC50 - >50.0 mg/L; NOEC - 25.0 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
50 mg/L
EC10 or NOEC for freshwater algae:
25 mg/L

Additional information

Algal growth inhibition has been investigated in a 72 hour test according to OECD test methods. Dissolution/dispersion agents were used to increase concentration of the substance in the test medium. The EC50 values for inhibition of specific growth rate (ErC50) and yield (EyC50) being >50.0 mg/L. The NOEC for inhibition of specific growth rate (ErC0) was 25.0 mg/L and yield (EyC0) was 50.0 mg/L.

Tests with a structural analogue of the registered substance, a trimellitate ester with mixed C8 -C10 side chains, showed no toxicity when tested at, or above, the limits of water solubility.

In accordance with ECHA guidance on information requirements and chemical safety assessment, Chapter R.7b: Endpoint specific guidance, the ErC50 endpoint is used in PNEC derivation. This is because use of values based on biomass cannot be applied to an analysis of results from a system in exponential growth without logarithmic transformation.