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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:
Experimental schedule: 10/31/2008 - 11/08/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to the OECD Testing Guideline and GLP compliant. All validity criteria are fulfilled. (and no deviation)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
SAMPLING:
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 also taken from the control.
For sampling at the end of the test, the test medium of the treatment replicates was pooled.
The samples were analyzed immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTIONS:
The test medium with the loading rate of 100 mg/L was prepared prior to the start of the test as follows: 74.8 uL of the test item (corresponding to
113 mg due to the test item density of 1.506 g/mL) were dissolved as far as possible in 1078 mL of test water using intense stirring for 3 hours at room temperature in the dark in a completely filled and tightly closed stirring vessel. Following the stirring period of 3 hours, the test medium was incubated in a separating funnel for a equilibration period of 15 minutes. During this period, different phases (a layer of test water with a maximum amount of dissolved test item and a layer of undissolved test item) were observable and were separately let out through the stopcock of the separating funnel.

Then the following dilution was performed form the stock solution: Dilution 1:100, 1:32, 1:10 and 1:3.2

The test method is based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, (formerly Selenastrum capricornutum),
- Strain: Strain No. 61.81 SAG
- Source: the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Gottingen, Gottingen / Germany).
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the test
- 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 test. 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
Post exposure observation period:
None
Hardness:
24 mg/L as CaCO3
Test temperature:
The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C:
Temperature was maintained at 22ºC throughout the test.
pH:
The pH values of the control cultures were observed to increase from pH 8 at 0 hours to pH 9.3 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Control: 8.0 - 9.3
Test solution: 7.9- 9.3
see table in "results"
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
The following treatments were tested: Dilution 1:100, 1:32, 1:10, 1:3.2 and the loading rate of 100 mg/L.
Additionally, a control was tested in parallel (test water without test item). Nominal concentrations of the test item exceeding 100 mg/L were not tested in accordance with the test guidelines.
Details on test conditions:
TEST SYSTEM /STUDY DESIGN:
- Test vessel:50-mL Erlenmeyer flasks. Each test flask was completely filled with test medium to keep the air-space in the test flasks as small as possible
- Type : As the test item is volatile, a closed system was used
- Material, size, headspace, fill volume: 50-mL Erlenmeyer flasks
- Aeration: No
- Initial cells density: 10000 cells/mL, The initial cell density was selected according to the recommendations of the OECD test guideline
- Control end cells density: The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM).
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): six replicates

Since the test item was determined to be volatile (according to the results of a pre-experiment, non-GLP), the test was performed in closed (glass-stoppered) 50-mL Erlenmeyer flasks. Each test flask was completely filled with test medium to keep the air-space in the test flasks as small as possible. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During the test, the test solutions were continuously stirred by magnetic stirrers.

A static test design was applied. The duration of the test was 72 hours.

TEST MEDIUM / WATER PARAMETERS:
As the test item is volatile, a closed system was used. Therefore, the algae were cultivated and tested in synthetic test water, prepared according to the test guidelines, but modified according to the International Standard ISO 14442 : the concentration of NaHCO3 was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer was added to keep the pH in the test media as constant as possible. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:

Ingredients Concentration
Macro-nutrients
NaHC03 250.0 mg/L
KH2PO4 1.6 mg/L
MgS04 x 7 H2O 15.0 mg/L
MgCl2 x 6 H20 12.0 mg/L
CaCl2 x 2 H20 18.0 mg/L
NH4CI 15.0 mg/L
HEPES-buffer

Trace elements
H3BO3 185.0 ug/L
MnCl2 x 4 H20 415.0 ug/L
ZnCl2 3.0 ug/L
CoCl2 x 6 H20 1.5 ug/L
CuCl2 x 2 H20 0.01 ug/L
Na2Mo04 x 2 H20 7.0 ug/L
FeCl3 x 6 H20 64.0 ug/L
Na2EDTA x 2 H20 100.0 ug/L

The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCOs).

- Culture medium different from test medium: yes
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and at the 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
- Adjustment of pH: No
- Temperature: The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C
- Photoperiod:
- Light intensity and quality: fluorescent tubes (Philips TLD 36W/840), approximately 7900 Lux (range: 7150 to 8570 Lux, measured at nine places in the experimental area)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter;The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800). The measurements were performed in duplicate.
- Sampling: A small volume of the algal suspension was daily withdrawn from each test flask for the measurement of the biomass, and was not replaced.
At the end of the test, a sample was taken from the control and from the test concentration of nominal 100 mg/L for visually inspection of the shape and size of the algal cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 , The enlarged spacing factor of 3.2 between the test concentrations was chosen, because according to the results of the range-finding test the concentration-effect relationship was rather flat and, thus, a large concentration range had to be tested.
- Test concentrations: Dilution 1:100, 1:32, 1:10, 1:3.2 and the loading rate of 100 mg/L.
- 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 optimum application method (non-GLP).
Reference substance (positive control):
yes
Remarks:
potassium dichromate
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.:
> 0.35 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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
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.:
> 0.35 mg/L
Nominal / measured:
meas. (geom. mean)
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:
EC50
Effect conc.:
> 0.35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
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.:
>= 0.35 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 : yes
- Observation of abnormalities: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.
- Any stimulation of growth found in any treatment: no


VERIFCATION OF TEST CONCENTRATIONS
At the start of the test, the analytically determined concentrations of the test item in the test media (dilution 1:3.2 and loading rate of 100 mg/L) were 0.52 and 1.9 mg/L, respectively (see Table 2 in "Any other information on results incl. tables". The test item concentrations for the dilutions 1:100, 1:32 and 1:10 could not be determined as they were below the limit of quantification (LOQ). 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 test item concentrations were below the LOQ in all samples.

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. As the concentrations at the end of the tests were below the LOQ of 0.134 mg test item/L, the geometric mean wascalculated from the concentrations at the start of the test and half the LOQ at the end of the test (i.e., 0.067 mg/L):
For the treatment Dilution 1:100, Dilution 1:32, Dilution 1:10 the mean measured concentration of the test item were below the LOQ, for the Dilution 1:3.2 the mean measured concentration of the test item was 0.19 mg/L and fot the loading rate of 100 mg/L mean measured concentration of the test item was 0.35 mg/L.


DATA EVALUATION:
The test item had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the loading rate of 100 mg/L (mean measured concentration: 0.35 mg/L; results of Dunnett's tests, one-sided, a = 0.05, Table 2 and Table 3 in "Any other information on results incl. tables").
The nominal test concentration of 100 mg/L (mean measured concentration: 0.35 mg/L) was, therefore, determined to be the 72-hour NOEC. This value might even be higher, but concentrations of the test item exceeding the loading rate of 100 mg/L were not tested. The 72-hour LOEC was higher than 100 mg/L (mean measured concentration: 0.35 mg/L).

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

At the start of the test, the pH measured in the treatments was between 7.9 and 8.0. At the end of the test, pH values of 8.9 to 9.3 were measured (Table 6). The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth despite the test media were stirred during the test. The water temperature during the test was 22 °C.
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 2008 showed that the sensitivity of the test system was within the historical range of Harlan Laboratories (72-hour EC50 for the growth rate: 1.20 mg/L (RCC Study No. B83755), range of the 72-hour EC50 for the growth rate from 2000 to 2008: 0.71-1.74 mg/L).
Reported statistics and error estimates:
The 72-hour EC10 and EC20 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis. however probit analysis were not relevant because of the low inhibitory effect of the test item on the algal growth at the tested concentrations.
The 72-hour EC50 values of the test item could not be calculated because of the low inhibitory effect of the test item on the algal growth at the tested concentrations.
For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Dunnett's tests

Table 1: Biomass of Algae

Treatment / Dilution

Mean measured

concentration

(mg/L)

Rep.

no.

Biomass of algae*

24 hours

48 hours 

72 hours

Control

 

 

 

 

1

2

3

4

5

6

4.4

3.1

3.8

3.8

2.9

4.2

17.1

16.4 17.3 16.8 14.3

16.1

122.7

120.9

113.1

113.1

104.1

110.9

Mean

SD

3.7

0.6

16.3

1.1

114.1

6.8

1:100

 

 

<LOQ

 

 

1

2

3

3.9

3.8

5.1

16.1

14.2

21.7

119.2

104.3

123.6

Mean SD

4.3

0.7

17.3

3.9

115.7

10.1

1:32

 

 

<LOQ

 

 

1

2

3

2.9

3.6

3.5

18.3

14.9 14.6

117.4

106.1

118.4

Mean

SD

3.3

0.4

15.9

2.1

114.0

6.8

1:10

 

 

<LOQ

 

 

1

2

3

4.0

3.5

4.1

14.3

17.5 16.0

108.1

116.6

121.4

Mean

SD

3.9

0.3

15.9

1.6

115.4

6.7

1:3.2

 

 

0.19

 

 

1

2

3

4.2

3.7

3.6

19.4

20.9

14.6

124.4

127.3

101.5

Mean

SD

3.8

0.3

18.3

3.3

117.7

14.2

100

 

 

0.35

 

 

1

2

3

3.5

3.2

3.0

14.8

13.9

14.4

107.5

89.9

119.0

Mean

SD

3.3

0.3

14.4

0.5

105.4

14.6

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 0.8 x 103relative fluorescence units.

Table 2     Average Growth Rates (µ)

Nominal

test item

concentration

(mg/L)

Mean

measured

concentration

(mg/L)

Average growth rate µ (day1) and inhibition of µ (Ir)

0-24 h

0-48 h

0-72 h

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

Control

1:100

1:32

1:10

1:3.2

100

----

<LOQ

<LOQ

<LOQ

0.19

0.35

1.51

1.65

1.4 0

1.56

1.54

1.38

0.0

-9.3 6.8 -3.5 -2.4

8.1

1.5 1.5 1.4 1.4 1.5 1.44

0.0

-1.5

1.0

0.9

-3.5

4.3

1.65

1.65

1.65

1.65

1.66

1.62

0.0

-0.3

0.0

-0.2

-0.6

1.7

Table 3      Yield (Y)

Nominal

test item

concentration

(mg/L)

Mean

measured

concentration

(mg/L)

Yield Y (x 103) and inhibition of Y (Iy)

 

 

0-24 h

0-48h

0-72h

 

 

Y

Iy (%)

Y

Iy (%)

Y

Iy

(%)

Control

1:100

 1:32

 1:10

1:3.2

100

------

<LOQ

<LOQ <LOQ

0.19

0.35

2.9

3.4

2.5

3.1

3.0

2.4

0.0

-19.0

13.2

-5.7

-3.5

15.7

15.5

16.5

15.1

15.1

17.5

13.5

0.0

-6.4

2.7

2.7

-12.7

12.8

113.3

114.9

113.2

114.6

116.9

104.6

0.0

-1.4

0.1

-1.1

-3.2

7.7

Table 4     Results Obtained for the Concentrations of the Treatment Samples

Age (hours)

Loading rate of 100 mg test

item/L

Measured concentration of test item

Sample preparation

factor F

Determined average concentration of

test item c (mg/L)

 

 

Sample 1

(mg/L)

Sample 2

(mg/L)

Average

(mg/L)

 

 

0

Control

Dilution 1:100

Dilution 1:32

Dilution 1:10

Dilution 1:3.2

Undiluted

<LOQana

<LOQana

<LOQana

<LOQana

0.435

0.886

---

<LOQana

<LOQana

<LOQana

0.584

2.80

n.a.

n.a.

n.a.

n.a.

0.510

1.84

1.01

1.01

1.01

1.01

1.01

1.01

n.a.

n.a

.n.a.

n.a.

0.515

1.86

72

Control

Dilution 1:100

Dilution 1:32

Dilution 1:10

Dilution 1:3.2

Undiluted

<LOQana

<LOQana

<LOQana

<LOQana

<LOQana

<LOQana

<LOQana

<LOQana

<LOQana

<LOQan

a<LOQana

<LOQana

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

1.01

1.01

1.01

1.01

1.01

1.01

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

LOQ = 0.134 mg test item/L

n.a.  =  not applicable

Validity criteria fulfilled:
yes
Remarks:
according to the criteria of the OECD TG 201
Conclusions:
The results obtained with loading rates were as follows:
72h-ErL50 > 100 mg/L (growth rate)
72h-EbL50 > 100 mg/L (yield)
72h-NOELR ≥ 100 mg/L (growth rate and yield)

The results obtained with the geometric mean of the measured concentrations were as follows:
72h-ErC50 > 0.35 mg/L (growth rate)
72h-EbC50 > 0.35 mg/L (yield)
72h-NOEC ≥ 0.35 mg/L (growth rate and yield)





Executive summary:

The effect of the test item 1,6-Divinylperfluorohexane on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72hour static test according to OECD Guideline 201 (2006), and the method C.3. of Commission Regulation (EC) No 440/2008. The study was compliant with the GLP.

Pre-experiments (non-GLP) were performed in order to determine the volatility, the solubility in test medium and the optimum application method. Thetest item 1,6-Divinylperfluorohexane was determined to volatile and poorly soluble in the test medium.

Since the test item is volatile, the test was performed in closed system i.e. the Erlenmeyer flasks were completely filled with test medium to keep the air-space in the test flasks as small as possible and modified and adapted test medium was used (according to the ISO 14442).

Since the test item is poorly soluble, the test was performed using the loading rate (according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), all the test solutions were prepared from a stock solution with a loading rate of 100 mg/L.

 

Based on results of a pre-experiment and on the results of a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to the following treatments: Dilution 1:100, 1:32, 1:10, 1:3.2 and the loading rate of 100 mg/L (three replicate flasks per concentration) and a control (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 22°C.

Samples of the algal populations were removed daily and Algal biomass determined for each control and treatment group, using afluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800).

 

Analysis of the test solutions at 0 hours showed measured test concentrations of 0.52 and 1.9 mg/L for the dilution 1:3.2 and loading rate of 100 mg/L respectively. The test item concentrations for the dilutions 1:100, 1:32 and 1:10 could not be determined as they were below the limit of quantification (LOQ).

Analysis of the test solutions at 72 hours showed a decrease of test item concentration in the test media since at the end of the test, the test item concentrations were below the LOQ in all samples.

Current regulatory advice is that in cases where a decrease in measured concentrations is observed, geometric mean measured concentrations should be used for determining the ECx and NOEC values. Therefore the reported 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 and to the loading rate of 100 mg/L. As the concentrations at the end of the test were below the LOQ of 0.134 mg test item/L, the geometric mean was calculated from the concentrations at the start of the test and half the LOQ at the end of the test (i.e., 0.067 mg/L): For the test solutions Dilution 1:100, Dilution 1:32, Dilution 1:10 the mean measured concentration of the test item were below the LOQ, for the Dilution 1:3.2 and the loading rate of 100 mg/L the mean measured concentration of the test item was 0.19 mg/L was 0.35 mg/L respectively.

No significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours at all the test concentration tested. The nominal test concentration of 100 mg/L (loading rate of 100 mg/L and geometric mean measured concentration: 0.35 mg/L) was, therefore, determined to be the 72-hour NOEC.

In addition, the microscopic examination (shape and size) 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 results obtained with loading rates were as follows:

72h-ErL50 > 100 mg/L (growth rate)

72h-EbL50 > 100 mg/L (yield)

72h-NOELR ≥ 100 mg/L (growth rate and yield)

The results obtained with the geometric mean of the measured concentrations were as follows:

72h-ErC50 > 0.35 mg/L (growth rate)

72h-EbC50 > 0.35 mg/L (yield)

72h-NOEC ≥ 0.35 mg/L (growth rate and yield)

 

It could be concluded that no toxicity was observed at the limit of solubility in the test medium. Limit of solubility in this test medium is 1.9 mg/L (result of the T0 analytical measurement of the stock solution of a loading rate of 100 mg/L).

 

The three validity criteria of the OECD guideline 201 were fulfilled, therefore this study is considered as reliable without restrictions.

 

According to the Guidance on the Application of the CLP Criteria, as the toxicity of the1,6-Divinylperfluorohexane is above the limit of the solubility in the test medium and as the best technic was used to achieve the maximum dissolved concentrations, 1,6-Divinylperfluorohexane should not be classified in accordance with the Regulation (EC) No 1272/2008.

 

 

 

Description of key information

According to the results of the study compliant with the GLP and with the OECD TG 201, the 1,6-Divinylperfluorohexane is considered as not toxic for the aquatic organisms at its limit of solubility. Therefore 1,6-Divinylperfluorohexane is not classified for the environmental part according to the Regulation (EC) 1272/2008.

Key value for chemical safety assessment

Additional information

One reliable key study is available for this endpoint (S.Hoger , 2010). In this study, the effect of the test item 1,6-Divinylperfluorohexane on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72hour static test according to OECD Guideline 201 (2006), and the method C.3. of Commission Regulation (EC) No 440/2008. The study was compliant with the GLP.

 

Following pre-experiments (non GLP), the test item 1,6-Divinylperfluorohexane was determined to be volatile and poorly soluble in the test medium, these pre-experiments were also performed to determine the optimum application method.

Therefore based on results of a pre-experiments and a preliminary range-finding test, a test was performed in closed system (i.e. the Erlenmeyer flasks were completely filled with test medium to keep the air-space in the test flasks as small as possible and modified and adapted test medium was used due to its volatility and the test solutions were prepared from a stock solution with a loading rate of 100 mg/L due to its poor solubility.

 

Pseudokirchneriella subcapitata was exposed to the following treatments: Dilution 1:100, 1:32, 1:10, 1:3.2 and the loading rate of 100 mg/L (three replicate flasks per concentration) and a control (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 22°C.

 

Samples of the algal populations were removed daily and Algal biomass determined for each control and treatment group, using afluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800).

 

The measured concentrations of the test solutions at T0 hours were 0.52 and 1.9 mg/L for the dilution 1:3.2 and loading rate of 100 mg/L respectively and below the limit of quantification (LOQ) for the dilutions 1:100, 1:32 and 1:10. The measured concentrations of the test solutions at T= 72 hours showed a decrease of test item concentration in the test media since at the end of the test, the test item concentrations were below the LOQ in all samples.

No significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours at all the test concentration tested.

 

The 72-hour NOEC (growth rate and yield) was therefore determined to be at least the highest test concentration. The reported 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 As the concentrations at the end of the test were below the LOQ of 0.134 mg test item/L, the geometric mean was calculated from the concentrations at the start of the test and half the LOQ at the end of the test.

 

The results obtained with loading rateswere as follows:

- 72h-ErL50 > 100 mg/L (growth rate)

- 72h-EbL50 > 100 mg/L (yield)

- 72h-NOELR ≥ 100 mg/L (growth rate and yield)

 

The results obtained with thegeometric mean of the measured concentrationswere as follows:

- 72h-ErC50 > 0.35 mg/L (growth rate)

- 72h-EbC50 > 0.35 mg/L (yield)

- 72h-NOEC ≥ 0.35 mg/L (growth rate and yield)

 

It could be concluded that no toxicity was observed at the limit of solubility of the test item 1,6-Divinylperfluorohexane in the test medium.