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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2008.08.29 to 2008.09.14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A decrease of test item concentration (between nominal and measured) in the test media was observed also at the start of the study. The mean measured test concentrations were between 15 and 72% of the nominal values. Results were based on samples which had been frozen and defrosted although some evidence is provided that the relative impact of this on the study should be low.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Directive 92/69/EEC 1992
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2008-11-12
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point:no data
- Boiling point: no data
- Vapour pressure: no data
- Water solubility (under test conditions): no data
- Henry's law constant: no data
- log Pow: no data
- pKa: no data
- Stability in water: no data
- Stability in light: no data
- pH dependance on stability: no data

OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: no data
- Other: The test item was determined to be volatile (according to the results of a pre-experiment performed without GLP).
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal concentrations = 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 mg/l and Mean measured concentrations = n.a., 0.032, 0.19, 0.72, 1.4, 3.0 mg/l. See table 1 in "any other information on materials and methods incl. tables" field.
- Sampling method: For measurement of the actual concentrations of the test item, duplicates samples (10 ml) were taken of all test concentrations at Time 0 and after 72h of exposure. At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled. The concentrations of the test item tert-Butyl-peroxybenzoate were determined in the duplicate test medium samples from the nominal test concentrations of 0.22 to 4.6 mg/l. The samples from the nominal test concentration of 0.10 mg/l were not analysed since this concentration was below the NOEC determined in this test. From the control samples, one of the duplicate samples was analysed from the corresponding sampling times.
- Sample storage conditions before analysis: All samples were deep-frozen immediately after sampling and stored at about -20°C until analysis (protected from light until analysis).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of the nominal concentration of 46 mg/l was prepared by dissolving 47.9mg of the test item completely in 1042ml of test water using intense stirring for 3 hours at room temperature in the dark. For this a tightly closed glass vessel with a minimum of air space was used to avoid evaporation of the test item. After stirring, the stock solution was diluted in a series of subsequent dilution steps with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
- Eluate:no data
- Differential loading: no data
- Controls: Test water without test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: formerly Selenastrum capricornutum
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): SAG, Institute for Plant Physiology, University of Göttingen, Göttingen / Germany
- Age of inoculum (at test initiation): The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
- Method of cultivation:An inoculum culture was set up four days before the start of the test.The algae were cultivated under the test conditions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
The water hardness (calculated) of the test water was 0.24 mmol/L (24 mg/L as CaCO3).
Test temperature:
Water Temperature during the Test Period
Day 0 (Start): 23°C
Day 1: 22°C
Day 2: 22°C
Day 3 (End) : 22°C
pH:
See Table 2 in the "Any information on materials and methods incl. tables" rich field.
Dissolved oxygen:
no data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 mg/L
Mean measured concentration of the test item: n.a., 0.032, 0.19, 0.72, 1.4, 3.0 mg/L. A decrease of test item concentration in the test media was observed. Already at the start of the study measured concentrations were lower than the nominals (83-87%) except in one case (107%). The mean measured test concentrations were between 15 and 72% of the nominal values. It is not certain therefore, whether the initial loss and final values are due to degradation of the test substance during the freeze-thaw process of the samlpes or due to loss during the preparation.
See table 1 in "Any other information on materials and methods incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyers flasks, closed system because the test item was determined to be volatile.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 ml erlenmeyer flasks completely filled with test medium to keep the air space as small as possible.
- Aeration: no data
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 1.0 x 10^4 cells/mL
- Control end cells density: not specified
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes, according to the test guidelines but modified according to the International Standard ISO 14442.
- Detailed composition if non-standard medium was used: The concentration of NaHCO3 was increased by 200mg/L to 250mg/L (as carbon source for the algal growth), and 6mmol/L HEPES-buffer were added to keep the pH in the test media as constant as possible.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: analytical grade salts were dissolved in sterile purified water
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data
- Other : no remarkable observations of the test media : clear test medium.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: The mean measured light intensity at the level of the test solutions was approximately 7600 Lux (range: 7150 to 8350 Lux, measured at nine places in the experimental area).
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter Model ZM). The algal biomass in the samples was determinated by fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800) at least in duplicate.
- Chlorophyll measurement: no data
- Other: none

TEST CONCENTRATIONS
- Spacing factor for test concentrations: about 2
- Justification for using less concentrations than requested by guideline: no data
- Range finding study : yes.
- Test concentrations in the range finding study: 0, 0.221 and 5.15 mg/L (nominal concentrations).
- Results used to determine the conditions for the definitive study: The selection of the test concentrations was based on the results of a range finding test and on results of a pre-experiment to determine the water solubility of the test item in the test water (non-GLP).
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI = 0.10-0.64 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CI = 0.13-0.20 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.54 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI = 0.18-0.75 mg/l
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CI = 0.19-0.26 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI = 0.48-1.12 mg /L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CI = 0.35-0.45 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.032 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): no data
- Observation of abnormalities (for algal test): yes
- Unusual cell shape: The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Colour differences: no data
- Flocculation: no data
- Adherence to test vessels: no data
- Aggregation of algal cells: no data
- Other: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 1.0mg/L and the algal cells in the control.
- Any stimulation of growth found in any treatment: slight stimulation at 0.22 mg/l at day 3.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium bichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in 2008 showed that the sensitivity of the test system was within the historical range of Harlan Laboratories (72-hour ErC50 = 1.20mg/l (RCC Study No. B83755), range of the 72-hour ErC50 from 2000 to 2008 = 0.71-1.74mg/l).
Reported statistics and error estimates:
The average growth rate of the algae after the test period of 72hours (see table 4 in "any other information on results incl. tables") was statistically significantly reduced at the test concentration of 1.4mg/L and at all higher test concentrations (results of Dunnett's tests, one-sided, alpha = 0.05).

Table 3: Biomass of algae

Nominal test item concentration (mg/L)

Mean measured concentration (mg/L)

Rep. no.

Biomass of algae*

24 H

48 H

72 H

Control

---

1

2

3

4

5

6

Mean

SD

4.6

4.2

4.0

4.1

4.1

4.2

4.2

0.2

19.4

18.4

16.4

17.2

17.9

20.8

18.4

1.6

117.8

117.4

119.7

131.9

122.8

145.9

125.9

11.2

0.10

---

1

2

3

Mean

SD

4.5

4.0

4.0

4.2

0.3

19.5

15.5

18.2

17.7

2.1

122.9

112.9

141.9

125.9

14.7

0.22

0.032

1

2

3

Mean

SD

3.9

3.6

4.3

3.9

0.4

17.9

16.7

16.9

17.2

0.7

132.8

136.1

133.6

134.2

1.7

0.46

0.19

1

2

3

Mean

SD

4.3

3.4

3.8

3.8

0.4

17.7

14.9

14.9

15.8

1.6

119.3

113.8

90.9

108.0

15.1

1.0

0.72

1

2

3

Mean

SD

2.7

2.5

2.8

2.7

0.2

8.0

8.7

9.4

8.7

0.7

25.8

28.2

26.4

26.8

1.2

2.2

1.40

1

2

3

Mean

SD

1.8

1.9

2.2

2.0

0.2

1.6

1.6

1.6

1.6

0.0

1.2

0.8

1.0

1.0

0.2

4.6

3.0

1

2

3

Mean

SD

1.5

1.4

1.4

1.4

0.1

0.5

0.5

0.4

0.5

0.1

0.4

-0.4

0.1

0.0

0.4

SD = Standard deviation

* The biomass was determined by fluorescent measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 10^3). At the start of the test, the initial cell density was 10000 algal cells/ml, corresponding to 1.1 x 10^3 relative fluorescence units.

Table 4: Average growth rates (µ)

Nominal test item concentration (mg/L)

Mean measured concentration (mg/L)

Average growth rate µ (day -1) and inhibition of µ (Ir)

0-24h

0-48h

0-72h

µ

Ir (%)

µ

Ir (%)

µ

Ir (%)

Control

0.10

0.22

0.46

1.0

2.2

4.6

--

--

0.032

0.19

0.72

1.4

3.0

1.37

1.37

1.30

1.28

0.92*

0.62*

0.28*

0.0

0.2

5.3

7.0

32.8

55.1

79.4

1.42

1.41

1.39

1.35

1.05*

0.21*

-0.40*

0.0

1.2

2.2

5.2

26.3

85.2

127.9

1.59

1.59

1.61

1.54

1.08

-0.02*

-1.45*

0.0

0.0

-1.4

3.3

32.4

101.6

191.0

* Mean value significantly lower than in the control (according to Dunnett's-tests, one-sided, alpha = 0.05)

Validity criteria fulfilled:
yes
Remarks:
All the validity criterion were fulfilled
Conclusions:
A study was realised in agreement with OECD Guideline 201 and all the validity criteria were all fulfilled. The ErC50 (72h)= 0.80 mg/L tert-Butyl-peroxybenzoate and a NOEC (72h) = 0.72 mg/L.
Executive summary:

The influence of the test item tert-Butyl-peroxybenzoate on the growth of the freshwater green algal species Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum (initial cell concentration = 1 x 10^4 cells/mL) was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006) and the EU Commission Directive 92/69/EEC, C.3 (1992). The nominal concentrations of the test item of 0.10, 0.22, 0.46, 1.0, 2.2 and 4.6 mg/L were tested in parallel with a control. The number of replicates per concentration was 3 and 6 per control. The light intensity was approximately 7600 Lux; the temperature was measured every days (22 -23°C); the pH was measured at day 0 and 3, it increased from 8.0 to 8.8. The algae was cultivated and tested in synthetic test water, prepared according to the test guidelines, but modified according to the ISO 14442. The algal biomass was determined by fluorescent measurement. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 15 and 72% of the nominal values. All results are based on measured test concentrations. The average growth rate of the algae after the test period of 72 hours was statistically significantly reduced at the test concentration of 1.4mg/L and at all higher test concentrations (results of a Dunnett's test). The 72 hours ErC50 = 0.80 mg/L with 95% Confidence Interval = 0.48 - 1.12 mg/L and the NOECr= 0.72 mg/l.

Description of key information

The 72h-ErC50 for algae based on geomean measured concentrations is 0.8 mg/L, the ErC10 is 0.44 mg/L (geomean measured).

Key value for chemical safety assessment

EC50 for freshwater algae:
0.8 mg/L
EC10 or NOEC for freshwater algae:
0.44 mg/L

Additional information

Two studies on algae are available. One study on freshwater algae performed under GLP with measured concentrations of the parent compound is considered reliable.

The second study based on nominal concentrations supports this conclusion as the ErC50 was higher than that based on measured concentrations.