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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:
08 Nov - 10 Dec 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 124691-59B
- Physical state: White solid
- Analytical purity: 97.6 %
- Expiration date of the lot/batch: 01 July 2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: Stable
Analytical monitoring:
yes
Details on sampling:

- Concentrations: All concentrations monitored
- Sampling method: One 2-mL sample was taken from all test concentrations and the control at times zero, 24h and 72h. One sample was held in reserve. A separate vessel was made for analytical sampling. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored frozen
- Abiotic samples: To verify maintanence of test substance concentration, a test vessel was set up at the highest substance concentration but without algae. Samples for analysis were taken at times zero, 24h and 72h.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a concentration of 100 mg/L applying an overnight period of magnetic stirring to accelerate the dissolution of test substance in medium. The lower concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none, test solutions were clear and colorless at start of test
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture

- Method of cultivation: Stock cultures were maintained in M1 medium (Nederlandse Praktijk Richtlijn no. 6505).
NaNO3, 500 mg/l
K2HPO4∙3H2O, 52 mg/l
MgSO4∙7H2O, 75 mg/l
Na2CO3∙10H2O, 54 mg/l
C6H8O7∙H2O, 6 mg/l
NH4NO3, 330 mg/l
CaCl2∙2H2O, 35 mg/l
C6H5FeO7∙xH2O, 6 mg/l
H3BO3, 2∙9 mg/l
MnCl2∙4H2O, 1.81 mg/l
ZnCl2, 0.11 mg/l
CuSO4∙5H2O, 0.08 mg/l
(NH4)6Mo7O24∙4H2O, 0.018 mg/l

ACCLIMATION
Four days before the start of the test, cells from the algal stock culture were inoculated in OECD medium at a cell density of 1e+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
22.1 - 24.0 °C
pH:
7.8 - 8.2
Nominal and measured concentrations:
Nominal: 0 mg/L, 10 mg/L, 18 mg/L, 32 mg/L, 56 mg/L, 100 mg/L
Measured: see Table 1
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass. Fill volume was 50 mL
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 124.4E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
1 replicate of the highest concentrations without algae
1 or 2 replicates of each test concentration for sampling

GROWTH MEDIUM
- Standard medium used: Yes - OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water (tap water purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges)
- Culture medium different from test medium: yes, propagation in M1, but a pre-culture was established in OECD medium 4 days before test initiation to use in test
- Intervals of water quality measurement: pH at beginning and 72 hours, temperature continuously

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: continuous illumination using 30 Watt 'Cool-white' TLD-Iamps. Light intensity within the range of 88 to 95 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beginning of the test, cells were counted with a microscope and counting chamber. Thereafter cell densities were measured using a spectrophotometer with immersion probe (pathlength = 10 mm). At the end of the study final microscopic observations were made on the highest test concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: Yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
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
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 13-28 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 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. The mean CV for section specific growth rates in the control was 18%, and the CV of average specific growth rates during the whole test period in control was 3%.
- Observation of abnormalities (for algal test): None observed. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to highest concentration when compared to the control.
- 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: yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 1.4 mg/L. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L.
- Other: Reference substance toxicity assay conducted 77 days before the test substance assay.
Reported statistics and error estimates:
Growth rate data were normally distributed by Chi-squared test, p = 0.4231 > 0.01. Variance of growth rate data were homogeneous by Levene's test, p(F) = 0.7579 > 0.01. NOEC was determined using Bonferroni t-test (α = 0.05). Calculation of the EC10 value was based on log-linear regression analysis of the percentages of growth rate reduction versus the logarithms of the corresponding nominal concentrations of the test substance. Statistical calculations were done in TOXSTAT Release 3.5, 1996.

Mean cell densities (x 1e+04 cells/mL)

Nominal conc. (mg/L) 

Exposure time (hours)

 

 

 0

24

48

72 

 control

 1.0

7.0 

25.2 

124.4 

 10

 1.0

6.7 

26.3 

124.9 

 18

 1.0

6.7 

20.8 

85.9 

 32

 1.0

6.7 

16.9 

58.6 

 56

 1.0

7.4 

12.7 

24.8 

 100

 1.0

7.0 

11.5 

14.7 

Percentage reduction of growth rate at different time intervals

Nominal conc. (mg/L)   

Mean growth rate

.

 µ (0 -24 h)

Reduction (%) 

 µ (24 -48 h)

  Reduction (%)

 µ (48 -72 h)

  Reduction (%)

 control

0.08091 

 

0.05349 

 

0.06631 

 

 10

0.07919

2.1 

0.05696 

-6.5 

0.06487

2.2 

 18

0.07910 

2.2

0.04723

11.7 

0.05920 

10.7 

 32

0.07939 

1.9 

0.3819 

28.6 

0.05196 

21.6 

 56

0.08290 

-2.5 

0.02285 

57.3 

0.02779 

58.1 

 100

0.08102 

-0.1 

0.02006 

62.5 

0.01094 

83.5

Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by an average factor of >16 within 2 days, the mean CV for section specific growth rates in the control was < 35% (18%), and the CV of average specific growth rates during the whole test period in control was < 7% (3%).
Conclusions:
The 72-hour EC50 (growth rate) of FBSEE diol to Pseudokirchneriella subcapitata was beyond the range tested (nominal concentration of 100 mg/L). The corresponding 72-hour NOEC (growth rate) was 10 mg/L. Test conducted per OECD 201.
Executive summary:

The toxicity of FBSEE diol to the green algae, Pseudokirchneriella subcapitata, was assessed in a 72-hour toxicity test conducted according to the OECD 201 method. An exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met. Nominal concentrations were confirmed by analytical measurement (98-110%).

The 72-hour ErC50 of FBSEE diol to Pseudokirchneriella subcapitata for growth rate was beyond the range tested (nominal concentration of 100 mg/L). The corresponding 72-hour NOEC (growth rate) was 10 mg/L and the 72-hour ErC10 was 19 mg/L (95% CI: 13-28 mg/L).

The study was performed in accordance with internationally-accepted test guidelines under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20-June-2011 to 24-June-2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Physical state: white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: store away from heat
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Initially a 100 mg/l solution was prepared by dilutiing 0.180 grams of FBSEE diacid to 1.8 liters of test media. The 100 mg/L solution was stirred on a magnetic stir plate for two hours to ensure complete solubility of FBSEE diacid in the dilution water. The 100 mg/L solution was then used to prepare lesser test concentrations.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material: Visual observations of the test solutions did not show any color change or precipitates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata 22662
- Source (laboratory, culture collection): strain from American Type Culture Collection

ACCLIMATION
- Acclimation period: Algal stock was subcultured in OECD recommended medium for 8 days before test initiation
- Culturing media and conditions (same as test or not): Same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
23.1 - 23.4 °C
pH:
6.26 - 9.57
Conductivity:
195 - 216 µmhos/cm
Nominal and measured concentrations:
Nominal concentrations: 0 (dilution water control), 6.3 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with foam stoppers
- Material, size, headspace, fill volume: borosilicate glass, 250 mL with 100 mL fill volume
- Aeration: test vessels held on shaker table set at 100 rpm
- Initial cells density: 2.4E+04 cells/mL
- Control end cells density: 49.5E+04 cells/mL
- No. of vessels per concentration (replicates): four
- No. of vessels per control (replicates): four

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water
- Conductivity: 195 - 216 µmhos/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: At test initiation and termination - pH, conductivity and temperature; daily - temperature

OTHER TEST CONDITIONS
- Sterile test conditions: Test vessels and test medium were autoclaved
- Adjustment of pH: No
- Photoperiod: continuous illumination
- Light intensity and quality: 7920 to 8150 lux, using 30 Watt 'Cool-white' fluorescent Iamps

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Daily cells counts using direct microscopic enumeration via an Improved Neubauer hemacytometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 0 mg/L, 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
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
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
Details on results:
- Exponential growth in the control (for algal test): yes. The mean CV for section specific growth rates in the control was 6% for the 24 hour test interval and the 48-hour to 96-hour mean CVs ranged from 2-3%. The CV of average specific growth rates during the whole test period in the control was 2%.
- Observation of abnormalities (for algal test): not reported
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
Growth rate data were normally distributed by Shapiro - Wilks test, D = 0.0036; W = 0.9777. Variance of growth rate data were homogeneous by Bartlett's test, calculated B1 statistic = 4.2810; p-value = 0.5097. NOEC was determined using Dunnett's test (α = 0.05). The mean coefficient of variation for specific growth rates in control cultures for section-by-section was 6% for the 24-hour test interval. The 48-hour to 96-hour mean coefficient of variations ranged from 2-3%. The coefficient of variation of average specific growth rates in replicate control cultures was 2%. Statistical calculations were done using ICp software (EC10s and EC50s) and TOXSTAT Release 3.5, (NOECs and LOECs).

Mean cell densities (x 1E+04 cells/mL)

Nominal conc. (mg/L) 

 Exposure time (hours)    

   0 24   48 72 
 control  2.4  10.0  11.5  40.0
 6.3  2.4  9.0  13.5 41.3 
 12.5  2.4  9.0 12.3  39.3 
 25  2.4  9.0  13.0 36.5 
 50  2.4  7.8  11.8 38.5 

 100

 2.4  7.8  12.3 37.0 

Percentage reduction of growth rate at different time interval

Nominal conc. (mg/L)   Mean growth rate               
   µ (0 - 24 h)  Reduction (%)  µ (0 - 48 h)   Reduction (%)   µ (0 - 72 h)   Reduction (%)
control  1.427    0.783   0.938  
6.3  1.322 7.36 0.864  -10.34 0.948  -1.07
12.5  1.322 7.36  0.817  -4.34  0.932  0.64 
25  1.322 7.36   0.845  -7.92 0.907  3.30 
50  1.179 17.38  0.796  -1.66  0.925 1.39 
100  1.179 17.38  0.817  -4.34  0.912  2.77 
Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by a factor of 17 during the first 3 days, the mean CV for section specific growth rates in the control was < 35% (6%), and the CV of average specific growth rates during the whole test period in control was < 7% (2%).
Conclusions:
The 72-hour EC50 and EC10 (growth rate) of FBSEE diacid to Pseudokirchneriella subcapitata was beyond the range tested (nominal concentration of 100 mg/L). The test was conducted per OECD TG 201.
Executive summary:

FBSEE diacid is a degradation product of FBSEE diol. The toxicity of FBSEE diacid to the green algae,Pseudokirchneriella subcapitata, was assessed in a 72-hour toxicity test conducted according to the OECD 201 method. An exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met. The 72-hour EC50 and EC10 of FBSEE diacid to Pseudokirchneriella subcapitatafor growth rate were beyond the range tested (nominal concentration of 100 mg/L). The study followed an international standard method but was not GLP compliant. The results are for a degradation product rather than the parent material. Therefore the study is deemed reliable with restrictions.

Description of key information

The 72-hour EC50 (growth rate) of FBSEE diol to Pseudokirchneriella subcapitata was beyond the range tested (nominal concentration of 100 mg/L). The corresponding 72-hour EC10 (growth rate) was 19 mg/L. The test was conducted per OECD TG 201.

Key value for chemical safety assessment

Additional information

In the key study, the toxicity of FBSEE diol to the green algae, Pseudokirchneriella subcapitata, was assessed in a 72-hour toxicity test conducted according to the OECD 201 method. An exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met. Nominal concentrations were confirmed by analytical measurement (98-110%). The 72-hour ErC50 of FBSEE diol to Pseudokirchneriella subcapitata for growth rate was beyond the range tested (nominal concentration of 100 mg/L). The corresponding 72-hour NOEC (growth rate) was 10 mg/L and the 72-hour ErC10 was 19 mg/L (95% CI: 13-28 mg/L). The study was performed in accordance with internationally-accepted test guidelines under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis

FBSEE diol will be rapidly converted to FBSEE diacid (see section 5.2.1). In a supporting study the toxicity of FBSEE diacid to the green algae, Pseudokirchneriella subcapitata, was assessed in a 72-hour toxicity test conducted according to the OECD 201 method. An exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met. The 72-hour EC50 and EC10 of FBSEE diacid to Pseudokirchneriella subcapitata for growth rate were beyond the range tested (nominal concentration of 100 mg/L). The study followed an international standard method but was not GLP compliant. The results are for a degradation product rather than the parent material. Therefore the study is deemed reliable with restrictions.