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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:
14 Apr - 12 Jun 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland
Analytical monitoring:
yes
Details on sampling:
For measurement of the actual concentrations of the test item, duplicate 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 taken from the control.For sampling at the end of the test, the test medium of the treatment replicates was pooled.
All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis.
In pre-experiments, the test item proved to be stable under these storage conditions.The concentrations of the test item Cyrene™ were determined in both of the duplicate test medium samples from the highest nominal test concentration of 100 mg/L. The samples from all lower nominal test concentrations were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed per sampling time.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 50.25 mg of the test item completely in 500 mL of test water under intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was serially diluted 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.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Common name: green alga- Strain: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG
- Source (laboratory, culture collection): Supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines. An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 °C
pH:
7.2 - 8.4
Nominal and measured concentrations:
Control, 6.25, 12.5, 25, 50 and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM

- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): closed; covered with a glass dish
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks containing 15 mL of test solution
- Stirring: continuous
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS

- Preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the respective concentrations (see "any other information on materials and methods" below)
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS

- Photoperiod: continuous
- Light intensity and quality: 6500 to 8760 Lux; fluorescent tubes (Philips TLD 36W-1/840)
- Temperature: Test flasks were incubated in a temperature-controlled water bath at a temperature of 24 °CEFFECT PARAMETERS MEASURED (with observation intervals if applicable): biomass, daily
- Determination of cell concentrations: fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm). The measurements were performed at least in duplicate. At the end of the test, a sample was taken from the control and from the test concentration of nominal 100 mg/L to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS

- Range finding 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
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
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
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
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:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
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 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. The pH rose in the control from 7.3 at test start to 8.4 at test end fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test.
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in March 2014 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.1 mg/L (Harlan Study Number D88947), range of the 72-hour EC50 for the growth rate from 2000 to 2014: 0.71 - 1.7 mg/L).
Reported statistics and error estimates:
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t test, one-sided smaller, alpha = 0.05 [Williams, 1971; Williams, 1972].WILLIAMS, D. A. (1971): A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27, 103-117.WILLIAMS, D. A. (1972): The comparison of several dose levels with a zero dose control. Biometrics 28, 519-531.

Analytical results:

The measured concentrations of Cyrene™ in the test medium with the test concentration of 100 mg/L were 103 and 95% of the nominal value at the start and at the end of the test, respectively. Thus, the test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item.

Reviewer's note: The sample has been directly injected in the GC-FID which would result in the anhydrous form of Cyrene™ being detected, however, the organisms would have been exposed to Gem Diol.

Table: Biomass of Algae

Nominal test item concentration [mg/L]

Rep. no.

Biomass of algae*

24 hours

48 hours

72 hours

Control

1

3.4

22.8

101.6

2

3.9

26.4

106.9

3

3.3

18.1

95.2

4

3.3

19.0

99.4

5

3.7

23.2

101.0

6

4.1

25.0

131.2

Mean

3.6

22.4

105.9

SD

0.3

3.3

13.0

6.25

1

3.6

25.9

128.6

2

3.8

24.6

106.8

3

4.1

25.0

121.4

Mean

3.8

25.2

119.0

SD

0.3

0.6

11.1

12.5

1

3.5

26.4

123.8

2

3.3

22.3

98.0

3

3.9

24.0

117.4

Mean

3.5

24.2

113.1

SD

0.3

2.1

13.5

25

1

2.8

20.5

103.8

2

3.6

25.7

109.0

3

3.1

19.8

107.4

Mean

3.2

22.0

106.7

SD

0.4

3.2

2.7

50

1

3.2

26.0

122.9

2

2.7

19.3

86.1

3

3.5

24.9

115.5

Mean

3.2

23.4

108.2

SD

0.4

3.6

19.5

100

1

2.6

20.4

106.4

2

2.4

19.7

94.1

3

2.4

19.6

90.3

Mean

2.5

19.9

96.9

SD

0.1

0.4

8.4

* The biomass was determined by fluorescence 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 5000 algal cells/mL, corresponding to 0.62 x 10^3 relative fluorescence units

Table: Average Growth Rates (µ)

Nominal test item concentration [mg/L]

Average growth rate µ/day and inhibition of µ (Ir)

0 – 24 h

 

 

0 – 48 h

 

 

0 – 72 h

 

 

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

1.760

0.0

1.790

0.0

1.712

0.0

6.25

1.814

-3.1

1.852

-3.5

1.751

-2.3

12.5

1.742

1.0

1.832

-2.3

1.734

-1.3

25

1.633*

7.2

1.782

0.5

1.716

-0.3

50

1.621*

7.9

1.811

-1.2

1.717

-0.3

100

1.378*

21.7

1.734

3.1

1.683

1.7

Table: Yield (Y)

Nominal test item concentration [mg/L]

Yield Y (x 10^3) and inhibition of Y (Iy)

0 – 24 h

0 – 48 h

0 – 72 h

Y

Iy [%]

Y

Iy [%]

Y

Iy [%]

Control

3.0

0.0

21.8

0.0

105.3

0.0

6.25

3.2

-6.4

24.6

-12.6

118.3

-12.4

12.5

2.9

2.2

23.6

-8.3

112.4

-6.8

25

2.6

14.2

21.4

2.0

106.1

-0.8

50

2.5*

15.5

22.8

-4.4

107.6

-2.2

100

1.8*

38.5

19.3

11.7

96.3

8.5

* mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, alpha = 0.05)

Table: Section-by-Section Growth Rates

Nominal test item concentration [mg/L]

Section-by-section growth rates (day-1) and inhibition of the growth rates (Ir)

0 – 24 h

24 – 48 h

48 – 72 h

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

1.76

0.0

1.82

0.0

1.56

0.0

6.25

1.81

-3.1

1.89

-3.9

1.55

0.4

12.5

1.74

1.0

1.92

-5.6

1.54

1.1

25

1.63

7.2

1.93

-6.1

1.59

-1.9

50

1.62

7.9

2.00

-10.0

1.53

1.8

100

1.38

21.7

2.09

-14.9

1.58

-1.7

Validity criteria fulfilled:
yes
Conclusions:
A 72 hour EC50 value of >100 mg/l and an EC10 of >100 (and NOEC ≥100 mg/l) (measured) (highest concentration tested) have been determined for the effects of Cyrene™ (CAS 53716-82-8) on growth rate of Pseudokirchneriella subcapitata based on mean measured GC concentrations.

In view of the properties of the substance, it is thought that the organisms were exposed to the Gem Diol form of the substance.

Refer to the discussion in IUCLID Section 6.0 for further discussion of the approach to chemical safety assessment for this registration substance.

Description of key information

Short-term toxicity to aquatic algae: 72 hour EC50 >100 mg/l and an EC10 >100 mg/l (and a NOEC of ≥100 mg/l (measured) (highest concentration tested) (OECD 201 (Algal Inhibition test)), tested on the registration substance related substance, (1S,5R)-6,8-dioxabicyclo[3.2.1]octan-4-one (CAS 53716-82-8).

Key value for chemical safety assessment

Additional information

A 72 hour EC50 value of >100 mg/l and an EC10 of >100 (and NOEC ≥100 mg/l) (measured) (highest concentration tested) have been determined for the effects of Cyrene™ (CAS 53716-82-8) on growth rate of Pseudokirchneriella subcapitata based on mean measured GC concentrations. The measured concentrations were within 80% of the nominal concentration, therefore the results are reported in terms of nominal concentration.

In view of the properties of the substance, it is thought that the organisms were exposed to the Gem Diol form of the substance.

Refer to the discussion in IUCLID Section 6.0 for further discussion of the approach to chemical safety assessment for this registration substance.