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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:
25 January - 2 August 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accomodated fractions (WAFs) were prepared. The test substance was added to test medium and stirred for 52 hrs. Vessels had minimal headspace to prevent volitilization. Contents of the vessels were then allowed to settle for 1-2 hrs. Aqueous phase was used for the test. Controls were subject to the same regime
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, Maryland, Ohio, U.S.A.
- Method of cultivation: Axenic stock cultures are maintained on agar plates. These are used to inoculate liquid cultures which, while in exponential phase growth, are in turn used to inoculate test media.Cells are cultured in as 100 mL batches in 240 mL Erlenmeyer flasks for up to 4 days. They are maintained at nominally 21-25°C in an orbital incubator under constant illumination. Algal cultures are streaked onto agara plates and maintained under continuous illumination in a vertical incubator. Cultures are renewed approximately every fortnight.


ACCLIMATION
- Culturing media and conditions (same as test or not): yes
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.7 - 24.2°C
pH:
8.3 - 9.1
Nominal and measured concentrations:
0, 1, 3, 10, 30, 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: Erlenmeyer flasks, 287 mL, no headspace
- Initial cells density: 5000 cells/mL (in 3 test flasks and 6 control flasks)
- Control end cells density: 0.095 million cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 7


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Nutrient medium was prepared by dissolving Analar grade salts in reverse osmosis filtered water
- Total organic carbon: 2.5 mg/L
- Chlorine: 0.02 mg/L Cl2
- Alkalinity: 257 mg/L CaCO3
- Ca/mg ratio: 48:1
- Conductivity: 496 µs/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: 6 months


OTHER TEST CONDITIONS
- Photoperiod: constant illumination
- Light intensity and quality: approx. 5200 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth rate
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Range finding study
- Results used to determine the conditions for the definitive study: 72h EL50: 1 -10 mg/L
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
10 - 30 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
biomass
Remarks on result:
other: time: 0-24 h
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
10 - 30 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other: time: 0-24 h
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
biomass
Remarks on result:
other: time: 0-24 h
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other: time: 0-24 h
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
10 - 30 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
biomass
Remarks on result:
other: time: 0-48 h
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
10 - 30 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other: time: 0-48 h
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
biomass
Remarks on result:
other: time: 0-48 h
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other: time: 0-48 h
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
10 - 30 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
biomass
Remarks on result:
other: time: 0-24 h
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
10 - 30 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other: time: 0-24 h
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
biomass
Remarks on result:
other: time: 0-24 h
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other: time: 0-72 h
Reported statistics and error estimates:
Loading rates used to produce WAF that cause a 50% effect (LL50) were estimated by visual inspection of the data. This was considered more appropriate than an interpolative statistical analysis when considering toxicity data based on WAF of test substances that are mixtures containing components of low solubility.

Table: Growth of Raphidocelis subcapitata cultures exposed to WAF of SBP 100/140

Loading rate used to produce WAF

Cell concentration (million cells/mL)

Mean cell concentration (million cells/mL)

Calculated mean area (A) under growth curve (+ 1 million)

Mean reduction in A relative to controls (%)

Mean calculated average specific growth rate (u)

Mean reduction in u relative to controls (%)

 

0h

24h

48h

72h

0h

24h

48h

72h

0-24h

0-48h

0-72h

0-24h

0-48h

0-72h

0-24h

0-48h

0-72h

0-24h

0-48h

0-72h

Control

0.0049

0.011

0.04

0.087

0.0054

0.013

0.043

0.095

0.086

0.62

2.1

 

 

 

0.035

0.043

0.04

 

 

 

 

0.0058

0.011

0.042

0.092

 

0.0049

0.013

0.042

0.095

 

0.0056

0.014

0.043

0.1

 

0.0055

0.014

0.045

0.097

 

0.0057

0.013

0.044

0.097

1

0.0047

0.01

0.036

0.061

0.0048

0.011

0.043

0.078

0.07

0.6

1.9

18

2.9

9.8

0.033

0.045

0.038

5.5

-5.6

3.7

 

0.0048

0.011

0.047

0.086

 

0.005

0.011

0.046

0.086

3

0.0048

0.012

0.042

0.083

0.0049

0.012

0.042

0.09

0.09

0.62

2.1

-4.6

-0.73

2.6

0.039

0.045

0.04

-10

-3.8

-1.3

 

0.0048

0.013

0.041

0.085

 

0.0051

0.012

0.043

0.1

10

0.005

0.0094

0.038

0.11

0.0051

0.0098

0.039

0.12

0.056

0.52

2.3

34

16

-8.4

0.027

0.042

0.044

22

2.2

-10

 

0.005

0.01

0.034

0.13

 

0.0053

0.01

0.044

0.13

30

0.0052

0.0065

0.009

0.0092

0.0049

0.0058

0.009

0.009

0.011

0.071

0.17

87

89

92

0.007

0.013

0.0084

80

71

79

 

0.0044

0.0053

0.0099

0.0099

 

0.0053

0.0057

0.0082

0.008

100

0.0051

0.0057

0.0081

0.011

0.0051

0.0054

0.006

0.0077

0.0037

0.018

0.059

96

97

97

0.0025

0.0025

0.0048

93

94

88

 

0.0053

0.0052

0.0054

0.0073

 

0.0049

0.0053

0.0044

0.0046
Validity criteria fulfilled:
yes
Conclusions:
This study examined the toxicity of hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics to algae. The algae Raphidocelis subcapitata was exposed to loading rates of 0, 1, 3, 10, 30, or 100 mg/L of test substance for 72 hrs. The test solution was not renewed and cell counts were taken at 0, 24, 48, and 72 hrs after start of exposure. No analytical monitoring was done as studies of closely related products has shown that the solubility of the test substance is likely to be less than the determination limit of the analytical equipment. The 72-hr EbL50 for algae was 10 -30 mg/L (WAF), and the 72-hr ErL50 was 10-30 mg/L (WAF). The 72-hr NOEL value for biomass and growth rate was 10 mg/L.
Executive summary:

This study examined the toxicity of hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics to algae. The algae Raphidocelis subcapitata was exposed to loading rates of 0, 1, 3, 10, 30, or 100 mg/L of test substance for 72 hrs. The test solution was not renewed and cell counts were taken at 0, 24, 48, and 72 hrs after start of exposure. No analytical monitoring was done as studies of closely related products has shown that the solubility of the test substance is likely to be less than the determination limit of the analytical equipment. The 72-hr EbL50 for algae was 10 -30 mg/L (WAF), and the 72-hr ErL50 was 10-30 mg/L (WAF). The 72-hr NOEL value for biomass and growth rate was 10 mg/L.

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:
other: Adopted according to OECD SIDS (public available peer reviewed source) and respective study summary. The original source is not available and has not been reviewed.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
GLP compliance:
yes
Analytical monitoring:
yes
Details on test solutions:
Individual treatment concentrations were prepared as water accommodated fractions (WAF). Control and dilution water were laboratory prepared algal nutrient medium. Test substance was mixed with dilution water in covered vessels for ~22 hours, and the mixtures were allowed to settle for ~21 hours prior to drawing off the aqueous phase for testing.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): Algal cells obtained from testing laboratory cultures. Original algal cultures obtained from University of Texas, Austin, Texas
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
23 - 26°C
pH:
7.7-8.1 at test initiation
8.1-10.1 at 72 hours
8.0-10.7 at test termination
Nominal and measured concentrations:
0, 3.1, 6.3, 13, 25, 50, and 100 mg/L (nominal)
Measured concentrations of the 0 and 96 hour samples ranged from 0.50 to 11% of nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 300 mL bottles that were completely filled with test solution
- Initial cells density: 5000 cells/mL
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

OTHER TEST CONDITIONS
- Photoperiod: 24 h
- Light intensity and quality: 4010-4730 lux
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
biomass
Remarks on result:
other: 95% Cl 9.8 to 16
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
29 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl 18 to 35
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
6.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
6.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
other: cell density
Remarks on result:
other: 95% Cl 11 to 14
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
6.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
other: cell density
Reported statistics and error estimates:
Linear interpolation (ICp) for EL50s. ANOVA and Dunnett’s test for NOELs.

The pH increased by more than one unit after 72 hours as a result of good algal growth in the cultures. This increase was not sufficient to invalidate the study.

Tab: Inhbition in %

Nominal concentration (mg/L)

% inhibition (72 h)

% inhibition (96 h)

0

-

-

3.1

22

2.4

6.3

6.3

7.7

13

54

53

25

86

89

50

100

100

100

94

90

Tab.: Cell density in cells/mL

Nominal concentration (mg/L)

concentration (72 h) (cells/mL)

concentration (96 h) (cells/mL)

0

43.4 x E4

9.2 x E5

3.1

34.0 x E4

9.0 x E5

6.3

40.6 x E4

8.5 x E5

13

20.0 x E4

4.3 x E5

25

6.13 x E4

1.1 x E5

50

0

0

100

27.4 x E3

8.8 x E5
Validity criteria fulfilled:
not specified
Conclusions:
The toxicity, as measured by biomass and growth rate to the green alga (Pseudokirchneriella subcapitata formerly Raphidocelis subcapitata) was evaluated in freshwater. Under the conditions of this study, the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, produced a significant effect on biomass at 13 mg/L, based on nominal loading of the test substance in water.
Executive summary:

The toxicity, as measured by biomass and growth rate to the green alga (Pseudokirchneriella subcapitata formerly Raphidocelis subcapitata) was evaluated in freshwater. Under the conditions of this study, the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, produced a significant effect on biomass at 13 mg/L, based on nominal loading of the test substance in water.

Description of key information

EL50 (72 h) = 10 mg/L growth rate (Raphidocelis)
EL50 (72 h) = 10 mg/L biomass (Raphidocelis)
NOELR (72 h) = 6.3 mg/L growth rate (Raphidocelis)
NOELR (72 h) = 6.3 mg/L biomass (Raphidocelis)
NOELR (72 h) < 1 mg/L biomass (Raphidocelis)

Key value for chemical safety assessment

Additional information

No studies are available on the toxicity to aquatic algae for hydrocarbons, C7-C9, isoalkanes. Based on analogue approach data are read-across to hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics. Two studies conducted according to OECD 201 guideline are available on the toxicity to aquatic algae for hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics (Shell 1995, 2000). The toxicity, as measured by inhibition of growth rate and biomass to Raphidocelis, was evaluated in freshwater by using water accomodated fractions (WAFs). In this methodology, different loadings of the substance are added to the test medium and equilibrium between the water and the hydrocarbons is achieved according to their specific water solubility. The hydrocarbon concentrations at equilibrium will be characteristic of the loading of the substance, and therefore toxicity values from WAF studies are expressed as effect loadings or lethal loadings (EL or LL), not as concentrations. These effect loadings are used to determine environmental classifications. The WAF methodology is widely accepted for the testing of complex hydrocarbon substances and other UVCBs, and it has been incorporated in different guidance documents, including the REACH guidance (see draft decision response letter in Section 13).

Under the conditions of these studies, the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics produced toxicity to Raphidocelis at 10-30 mg/L measured as growth inhibition and biomass (Shell 1995) and 29 mg/L and 12 mg/L measured as growth inhibition and biomass, respectively (Shell 2000), based on nominal loadings of the test substance in water. For growth inhibition and biomass the NOELR was determined to be 10 mg/L in the study by Shell (1995) and 6.3 mg/L according to Shell (2000).