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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:
other: see 'Remark'
Remarks:
The reliability rating is a 1 because the study followed a standard guideline, which describes a procedure designed to evaluate this endpoint, and the results were reviewed for reliability and assessed as valid. The study was also conducted under GLP.
Justification for type of information:
Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples of each treatment WAF and the control were taken on Day 0 prior to the addition
of algae and at termination (composite of replicates) with no headspace. The samples were
stored under refrigeration until analyzed.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Algal cells were obtained from in-house culture.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
Mean test temperature: 24.2°C.
pH:
The pH was 7.7 in all of the test solutions at test initiation and ranged from 7.8 to 8.8 at test termination
Salinity:
freshwater
Nominal and measured concentrations:
Due to the relatively complex nature and limited water solubility of the test substance, the following exceptions to the guideline apply for this study: The concentration of the test substance in solution was not determined prior to use. Test substance analysis was performed on samples of the WAFs at the start of the test (day 0) and at termination (day 3). The initial concentration of the test substance was not maintained at 80% in the 2.2 mg/L and 7.6 mg/L treatments throughout the test (this may be due to biological activity or physical processes in the test chambers). It was deemed more appropriate to prepare individual treatment solutions by adding the test substance to dilution water and removing the WAF of each mixture for testing than to prepare dilutions of a stock solution. None of the above exceptions are believed to have affected the outcome, integrity, or quality of the study.
Details on test conditions:
Individual Water Accommodated Fractions (WAF's) were prepared for each treatment. The test substance was added to 4.4-4.5 L of algal nutrient medium augmented with sodium bicarbonate in glass aspirator bottles (capacity 4.5 L). The solutions were mixed for approximately 23.5 hours using a 6.5% vortex (of the static liquid depth). The test solutions were removed through the outlet at the bottom of each mixing vessel into 12 replicates of 140 mL in 125 mL Erlenmeyer flasks (no headspace) containing two 14mm glass spheres to facilitate mixing.. The test chambers were inoculated with algae (1.0 x 104 cells/mL) and were sealed with ground glass stoppers. Cell density was determined using a hemacytometer. Three replicates were sacrificed daily for cell density determination. The test chambers were placed on shaker tables (110 rpm) to keep the algae in suspension. The test was performed under static conditions with no aeration. The algae was cultured in-house from 5 day old stock cultures in log phase growth.

Continuous light: intensity was 7840 to 8498 Lux.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
7.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI = 4.1 to >25
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
3.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI = 0.38 - 48
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.07 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.07 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Reported statistics and error estimates:
The EbL50, ErL50 and confidence intervals for inhibition of growth/growth rate slope were determined by a probit regression calculation of the probit of the growth inhibition/growth rate slope vs the log of the loading rate and associated confidence intervals based on the methods of (Finny, 1971). Calculations were based on the PROC PROBIT procedure in (SAS, 2002). The NOELR for the EbL50 and ErL50 was based on (Duncan's, 1975) Multiple Range test and (Dunnett's, 1964) test, determined from the GLM procedure of (SAS, 2002). The (Shapiro-Wilk, 1965) test for normality was used to test if the assumption of normality of the residuals was met; since the residuals were normally distributed the NOEC was based on the estimated values.

1. Finney, D.J. 1971. Probit Analysis, 3rd Edition, London: Cambridge University Press.
2. SAS Version 8, SAS Institute, Inc., Cary, NC. 2002.
3. Duncan, D.B. 1975, "t-Tests and Intervals for Comparisons Suggested by the Data", Biometrics, 31, 339-359.
4. Dunnett, C. 1964, "New Tables for Multiple Comparisons With A Control", Biometrics, Vol 20, No. 3, pg 482-491.
5. Shapiro, S.S. and Wilk, M.B. 1965,"n analysis of variance test for normality (complete samples)" Biometrika, 52, pg 591-611.
Effects on growth rate (r) based upon actual loading rates:
72 hr ErL50 (loading rate) = 7.9 mg/L (4.1 - >25* mg/L)
72 hr NOELR (loading rate) = 0.22 mg/L

Effects on biomass (b) based upon actual loading rates:
72 hr EbL50 (loading rate) = 3.8 mg/L (CNC)
72 hr NOELR (loading rate) = 0.22 mg/L

Effects on growth rate (r) based upon measured concentrations:
72 hr ErC50 (measured conc.) = 0.42 mg/L (0.38 - 48 mg/L)
72 hr NOEC (measured conc.) = 0.07 mg/L

Effects on biomass (b) based upon measured concentrations:
72 hr EbC50 (measured conc.) = 0.29 mg/L (<0.07* - >0.81* mg/L)
72 hr NOEC (measured conc.) = 0.07 mg/L

Values in parenthesis are 95% confidence intervals.
* Confidence interval exceeded the highest or lowest loading rate or concentration tested.
CNC = Could Not Calculate 95% confidence intervals.

The analytical method used was headspace gas chromatography with flame ionization detection.

Summary of In-Life observations - % Inhibition
Loading Rate Control 0.22 0.69 2.2 7.6 25
(mg/L)
Meas. Conc. 0 0.07 0.18 0.32 0.43 0.81
(mg/L)

Based on Growth Rate
72 hours n/a 0.7 11 29 48 99
Based on Biomass
72 hours n/a 0.9 35 62 80 98
Validity criteria fulfilled:
yes
Conclusions:
Effects on growth rate (r) based upon actual loading rates:
72 ErL50 = 7.9 mg/L

Effects on biomass (b) based upon actual loading rates:
72 hr EbL50 = 3.8 mg/L

The test substance is a complex mixture, therefore, the results of the study are reported based on the actual loading rates of the test substance in algal media.
Executive summary:

Hydrocarbons, C9, Aromatics is moderately acutely toxic to freshwater algae.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02/12/1993-04/23/1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to Official Jounal of the European Communities No. L133 Algal inhibition test.
Justification for type of information:
Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10, 3, 1, 0.4, 0.1 control mg/l
- Sample storage conditions before analysis: all samples were analyzed on the day taken
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test material and dilution water were stirred in sealed vessels for 24 hrs. The mixture was then allowed to settle for 1 hr. The aqueous phase was then drawn off and used as the test medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: axenic strain
- Source (laboratory, culture collection): American Type Culture Collection, Maryland, Ohio, USA
- Age of inoculum (at test initiation): in exponential growth phase
- Method of cultivation: 1.5% mass/volume agar is added to liquid medium and autoclaved. After cooling, the medium is added to 9 cm diameter sterile petri dishes. Cultures are streaked onto the plates and maintained at 18-22°C under continuous illumination for up to 2 weeks. Liquid medium cultures are made by tranferring cells on a sterile loop from the agar cultures. 100 ml batch cultures are grown in 250 ml Erlenmeyer flasks for up to 4 days maintained at 22-26°C under constant illumination of ~3000 lux in an incubator.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Not determined
Test temperature:
21.9-24.3°C
pH:
7.2-10.3
pH exceeded preferred range in some test vessels. The flasks with good algal growth showed a large increase of pH (up to +3 pH units). Since they showed good growth, the high pH does not invalidate the test.
Dissolved oxygen:
Not determined
Salinity:
freshwater
Nominal and measured concentrations:
Nominal: 10, 3, 1, 0.4, 0.1, control mg/l
Measured: 9.4, 2.8, 1.3, 1.4, <1.0, <0.5 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed - sealed with glass stoppers
- Material, size, headspace, fill volume: 300 ml Erlenmeyer flask completely filled
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): static
- Initial cells density: 5000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: Medium as described in EPA-600/9-78-01B except 105 µg boric acid, and 50 mg/l sodium bicarbonate. Prior to adding sodium bicarbonate, the medium was autoclaved at 1.0 kg/cm² for 15 min. After cooling, the sodium bircarbonate was added.
- Detailed composition if non-standard medium was used: algal nutrient medium augmented with sodium carbonate


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Millipore filtered water.

OTHER TEST CONDITIONS
- Light intensity and quality: ~3000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counted with Coulter Multisizer
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
other: ErL50
Effect conc.:
2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 2.6-3.3 mg/l
Key result
Duration:
72 h
Dose descriptor:
other: EbL50
Effect conc.:
2.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 2.3-3.0 mg/l
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate

 Summary of In-Life observations - % Inhibition                  

Loading Rate (mg/L)  Control   0.1 0.4 1 3 10
Based on Growth Rate 72 hrs  n/a 0  0  0  55  100
Based on Biomass 72 hrs   n/a  0  0  0  78  99
Validity criteria fulfilled:
yes
Conclusions:
The 72-hr ErL50 for R. subcapitata is 2.9 mg/L (WAF). The 72-hr EbL50 is 2.6 mg/L (WAF).
Executive summary:

This study was done to determine the acute toxicity of Hydrocarbons, C9, aromatics to the algae R. subcapitata. R. Subcapitata was exposed to 0, 0.1, 0.4, 1, 3, or 10 mg/L (WAF) of the test substance for 72 hrs. The mean cell concentration was measured at 0, 24, 48, and 72 hrs of exposure. The average specific growth rate, and the area under the growth curves was then calculated for the exposure period. Significant reduction in growth as compared to controls was seen in the 3 and 10 mg/l test concentrations. The 72 -hr ErL50 for R. subcapitata was calculated to be 2.9 mg/L (WAF), and the 72 -hr EbL50 is 2.6 mg/L (WAF).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was given a reliability rating of 1 because it followed a standard test guideline, which describes a procedure to evaluate this endpoint, and the results were reviewed for reliability and assessed as valid. The study was also conducted under GLP.
Justification for type of information:
Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
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 sealed vessels for approximately 24 hours, and the mixtures were allowed
to settle for approximately 1 hour prior to drawing off the aqueous phase for testing.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Algal cells obtained from testing laboratory cultures. Original algal cultures obtained from American Type Culture Collection (ATCC 22662).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Temperature was 22 to 24 Deg C.
pH:
The pH ranged from 7.2 to 7.7 at test initiation and 7.2 to 10.2 at test termination.
Dissolved oxygen:
Not applicable
Salinity:
freshwater
Nominal and measured concentrations:
Nominal loading rates were 0.1, 0.4, 1.0, 3.0, and 10 mg/L. Only the 0 and 72 hours samples from the 3.0 and 10 mg/L treatments were analyzed yielding mean measured concentrations of 3.2 and 8.2 mg/L, respectively.
Details on test conditions:
Lighting was continuous at approximately 3000 lux.

Test vessels were sealed 300 ml conical flasks that were completely filled with test solution. The treatment and control flasks were inoculated with algal cells to yield initial concentrations ranging from 3200 to 5000 cells/ml. Uninoculated flasks were used to determine particle counts without algal cells. Biomass was calculated as area under the growth curve.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 - < 3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 - < 3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
ca. 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
ca. 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
See Tables Below
Reported statistics and error estimates:
EL50 values estimated by visual inspection of data to determine range of loading rates, which bracketed a 50% reduction in biomass and growth rate. ANOVA and Williams test used to determine NOELs.

Nominal Loading 72 h 
(mg/L)  % Inhibition
   
Control N/A
0.1 -14.0
0.4 -27.0
1.0 -8.0
3.0 97.0
10.0 100.0

Nominal Loading Conc.  Conc. 
(mg/L)  0 hr (cells/mL) 72 hr (cells/mL)
     
Control 3.2xE3 3.7xE5
0.1 4.6xE3 4.2xE5
0.4 5.3xE3 4.7xE5
1.0 5.4xE3 4.0xE5
3.0 5.2xE3 9.3xE5
10.0 3.5xE3 0.0
Validity criteria fulfilled:
yes
Conclusions:
The hydrocarbons, C10, aromatics, >1% naphthalene, freshwater 72-hr EL50 value for Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) is 1.0 to 3.0 mg/L based on both growth rate and biomass. The hydrocarbons, C10, aromatics, >1% naphthalene, freshwater 72-hr NOELR (No Observed Effect Loading Rate) value for Pseudokirchneriella subcapitata is approximately 1.0 mg/L based on both growth rate and biomass.
Executive summary:

The hydrocarbons, C10, aromatics, >1% naphthalene, freshwater 72-hr EL50 value for Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) is 1.0 to 3.0 mg/L based on both growth rate and biomass. The hydrocarbons, C10, aromatics, >1% naphthalene, freshwater 72-hr NOELR (No Observed Effect Loading Rate) value for Pseudokirchneriella subcapitata is approximately 1.0 mg/L based on both growth rate and biomass.

Description of key information

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

 

The data used to characterize the acute aquatic toxicity of C9 aromatic hydrocarbons are from two short-term toxicity studies with Pseudokirchneriella subcapitata, that followed standard test guidelines. The results indicate that C9 aromatics hydrocarbons causes moderate acute toxicity to freshwater green algae at a range of 2.9 to ≥7.9 mg/L (growth rate), and 2.6 to ≥3.8 mg/L (biomass), based on nominal loading of the test substance in water, and ≥0.42 mg/L and ≥0.29 mg/ L, respectively, based on measured concentrations.

 

Water accommodated fractions of hydrocarbons, C10, aromatics, >1% naphthalene, produced a freshwater 72-hr EL50 value for Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) of 1.0 to 3.0 mg/L based on both growth rate and biomass. The 72-hr NOELR (No Observed Effect Loading Rate) value for Pseudokirchneriella subcapitata is approximately 1.0 mg/L based on both growth rate and biomass. In a supporting study, water accommodated fractions of hydrocarbons, C10, aromatics, >1% naphthalene, produced a freshwater 72-hr EL50 value for Pseudokirchneriella subcapitata (formerlySelenastrum capricornutum) of 11 mg/L and 8.5 mg/L based on growth rate and biomass, respectively. The 72-hr NOELR (No Observed Effect Loading Rate) value for Pseudokirchneriella subcapitata is 2.5 mg/L based on both growth rate and biomass. 

 

This data is used for read across to Hydrocarbons, C9-C10, aromatics, >1% Naphthalene.

Key value for chemical safety assessment

Additional information

The toxicity of C9 aromatics hydrocarbons as measured by biomass and growth rate to the green alga (Pseudokirchneriella subcapitata formerly Selenastrum capricornutum) was evaluated in freshwater. Under the conditions of the studies, C9 aromatic hydrocarbons produced toxicity based on inhibition of growth rate at 2.9 to ≥7.9, and reduction in biomass at a range of 2.6 to ≥3.8 mg/L, based on nominal loading of the test substance in water, and ≥0.42 mg/L and ≥0.29 mg/ L, respectively, based on measured concentrations.

 

Water accommodated fractions of hydrocarbons, C10, aromatics, >1% naphthalene, produced a freshwater 72-hr EL50 value for Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) of 1.0 to 3.0 mg/L based on both growth rate and biomass. The 72-hr NOELR (No Observed Effect Loading Rate) value for Pseudokirchneriella subcapitata is approximately 1.0 mg/L based on both growth rate and biomass.