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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 2008-05-28 to 2010-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted following OECD Guideline 201 under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 26 hPa (at 20°C)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 1.5, 4.7, 15, 47, and 150 mg/L
- Sampling method: Quadruplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For the 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses. Two additional flasks of test medium with 150 mg/L were incubated under test conditions for analytical measurement. Immediately after sampling, acetonitrile (1 mL acetonitrile per mL sample volume) was added to each sample in order to stabilize the latter during the storage period. Thereafter, two of the quadruplicate samples were stored in the refrigerator (at about 5 °C) and the other two were analyzed immediately after addition of acetonitrile. The concentrations of the test item Di-t-butyl peroxide were determined immediately after sampling in two of the quadruplicate samples from all test concentrations at the start of the test. The concentrations of the test item were determined in all quadruplicate test medium samples from all concentrations at the end of the test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
- Sample storage conditions before analysis: no storage period.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item (244.01 mg) was dissolved in acetonitrile and made up to the mark in a 10 mL volumetric flask to prepare a stock solution with a concentration of 24401 mg/L. An aliquot of 0.21 mL of the stock solution were made up to 10 mL with acetonitrile/water (v/v; 1/1) to obtain fortification solution 1 with a concentration of 512 mg/L of the test item. Another stock solution aliquot of 3.1 mL was made up to 10 mL with acetonitrile/water (v/v; 1/1) to obtain fortification solution 2 with a concentration of 7564 mg/L of the test item. Defined volumes of these fortification solutions were diluted with test water to obtain spiked samples with concentrations of 4.61 mg/L (from fortification solution 1) and 151 mg/L (from fortification solution 2). Sample aliquots of 10 mL were diluted with the same volume of acetonitrile giving a sample preparation factor of two. In addition, test water without the test item was analyzed after sample preparation (analytical blank). Aliquots of the final solutions were analyzed filtered (0.45 µm, PTFE) and non-filtered by HPLC with UV/VIS detection.
- Eluate: distilled
- Differential loading: cf. Metod description above
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (Göttingen, Germany).
- Age of inoculum (at test initiation): in the exponential growth phase
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines. An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes, in the same conditions.
- Any deformed or abnormal cells observed: none
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no data
Hardness:
0.24 mmol/L
Test temperature:
22°C
pH:
at the start of the test: 8.1-8.2
at the end of the test: 8.1-9.6
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
nominal conc.: 0, 1.5, 4.7, 15, 47, 150 mg/L
measured conc.: 0, 0, 1.94, 5.52, 16.5, 61.6 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 60 mL of test medium
- Aeration: no data
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: 10e4 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no data
- Photoperiod: no data
- Light intensity and quality: approximately 7600 lux (range: 6570 to 8550 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter: Coulter Conter, Model ZM
- Chlorophyll measurement: none
- Other: none

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 0, 1.5, 4.7, 15, 47, and 150 mg/L
- Results used to determine the conditions for the definitive study: The selection of the test concentrations was based on the results of range-finding tests (non-GLP).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 12-17
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
ca. 20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 18-22
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 33-40
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 9.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 5.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 3.8-7.0
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
ca. 7.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 5.9-9.4
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 13-18
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 3.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis. For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Williams tests and Welch-t tests.

Table 1: Biomass of algae

Nominal test item concentration (mg/L)

Mean measured concentration (mg/L)

Rep. no.

Biomass of algae*

24 hours

48 hours

72 hours

Control

Control

1

10.6

47.8

276.9

 

 

2

9.7

46.4

324.3

 

 

3

9.6

51.1

279.4

 

 

4

10.4

47.7

255.5

 

 

5

8.9

43.5

323.1

 

 

6

9.3

48.2

279.5

 

 

Mean

9.7

47.4

289.8

 

 

SD

0.6

2.5

27.8

1.5

1.2

1

10.2

48.4

247.9

 

 

2

9.7

40.2

303.2

 

 

3

9.0

54.3

305.9

 

 

Mean

9.6

47.6

285.7

 

 

SD

0.6

7.1

32.7

4.7

3.9

1

9.6

38.8

234.3

 

 

2

8.8

30.0

250.1

 

 

3

8.6

51.0

292.4

 

 

Mean

9.0

40.0

258.9

 

 

SD

0.5

10.6

30.0

15

9.6

1

8.7

34.6

206.8

 

 

2

7.7

30.6

233.7

 

 

3

7.6

41.1

213.8

 

 

Mean

8.0

35.5

218.1

 

 

SD

0.6

5.3

13.9

47

27

1

5.7

23.1

65.6

 

 

2

5.4

18.6

51.0

 

 

3

5.5

17.9

69.8

 

 

Mean

5.5

19.8

62.2

 

 

SD

0.2

2.8

9.9

150

98

1

3.5

3.1

4.9

 

 

2

3.4

4.1

3.4

 

 

3

3.5

3.0

2.3

 

 

Mean

3.5

3.4

3.5

 

 

SD

0.1

0.6

1.3

SD:          Standard deviation

*:             The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 103). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 2.60 x 103relative fluorescence units.

Table 2: Average growth rate (µ)

Nominal
test item concentration

Mean measured concentration

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

0-24 h

0-48 h

0-72 h

(mg/L)

(mg/L)

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

Control

---

1.32

0.0

1.45

0.0

1.57

0.0

1.5

1.2

1.30

1.1

1.45

0.1

1.56

0.3

4.7

3.9

1.24*

6.0

1.35

6.7

1.53

2.4

15

9.6

1.13*

14.7

1.30*

10.2

1.48**

6.0

47

27

0.76*

42.8

1.01*

30.2

1.05**

32.8

150

98

0.29*

78.0

0.12*

91.4

0.09**

94.5

*:          mean value statistically significant lower than in the control
(according to Williams-test, one-sided,
a= 0.05)

**:        mean value statistically significant lower than in the control
(according to Welch-t-test, one-sided,
a= 0.05)

Validity criteria fulfilled:
yes
Conclusions:
The influence of the test item di-t-butyl peroxide on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72‑hour static test according to OECD Guideline 201 (2006) and the EU Commission Directive 92/69/, C.3 (1992), under GLP. The nominal concentrations of the test item of 1.5, 4.7, 15, 47 and 150 mg/L were tested in parallel with a control.  Results were EC10(72h) = 15 mg/L; EC20(72h) = 20 mg/L; EC50(72h) = 36 mg/L; NOEC = 3.9 mg/L; and LOEC = 9.6 mg/L for growth rate. For Yield: EC10(72h) = 5.5 mg/L; EC20(72h) = 7.8 mg/L; EC50(72h) = 15 mg/L; NOEC = 1.2 mg/L; and LOEC = 3.9 mg/L.
Executive summary:

The influence of the test item di-t-butyl peroxide on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72‑hour static test according to OECD Guideline 201 (2006) and the EU Commission Directive 92/69/, C.3 (1992), under GLP. The nominal concentrations of the test item of 1.5, 4.7, 15, 47 and 150 mg/L were tested in parallel with a control. 

As the test item is a volatile substance, the test was performed using Erlenmeyer flasks completely filled with test medium that were tightly sealed with glass stoppers to avoid losses of test item. The measured concentrations of the test item in the test media of the nominal test concentrations of 1.5 to 150 mg/L were between 70 and 83% of the nominal values at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, the measured values for the lowest test concentration of 1.5 mg/L were below LOQ (Limit of quantification, LOQbio: 2.30 mg/L).For the other test concentrations, 46 to 84% of the nominal values were found at the end of the test.

The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and the end of the test:

 

Nominal test concentration

Mean measured concentration of the test item
(geometric mean)

(mg/L)

(mg/L)

(% of nominal)

1.5*

1.2

80

4.7

3.9

84

15

9.6

64

47

27

57

150

98

65

 

*for this concentration the average concentrations at the start and at the end of the test were

< LOQ(Limit of quantification, LOQbio: 2.3 mg/L); the1/2 LOQbio(1.15 mg/L) was used

as start and end value to calculate the geometric mean.

 

The biological results of the study were as follows (on the basis of mean measured concentrations of the test item):

 

Parameter

Growth rate

Yield

(0-72 h)

 

 

EC10  (mg/L)

15

5.5

95% confidence interval

12 – 17

3.8 – 7.0

EC20  (mg/L)

20

7.8

95% confidence interval

18 – 22

5.9 – 9.4

EC50  (mg/L)

36

15

95% confidence interval

33 – 40

13 – 18

NOEC (mg/L)

3.9

1.2

LOEC (mg/L)

9.6

3.9

 

Description of key information

A GLP study on Pseudokirchneriella subcapitata following OCED 201 conducted in sealed vessel with analysis was performed. The geometric mean of measured concentrations was used providing a 72h-ErC50 = 36 mg/L and a 72 h ErC10 of 15 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
36 mg/L
EC10 or NOEC for freshwater algae:
15 mg/L

Additional information

A reliable GLP study on Pseudokirchneriella subcapitata following the 2006 updated OCED 201 (Hoger, 2010). Due to the volatility of the substance, the test was conducted in sealed vessels and analytical measurements were made. Despite this, after 72 hours, concentrations of test groups decreased to 46%. Thus the geometric mean of measured concentrations at the test initiation and termination was calculated and a 72h-ErC50 = 36 mg/L was determined.