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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
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)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.201, March 23, 2006,Annex 5 corrected: July 28, 2011, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test"
Deviations:
no
Qualifier:
according to
Guideline:
other: "Algal Growth Inhibition Test" stipulated in the "Testing Methods for New Chemical Substances" March 31, 2011, No. 0331-7
Version / remarks:
"Algal Growth Inhibition Test" stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011, No. 0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No. 5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; April 2, 2012 partial revision, No. 0402-1, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 28, 2012, No. 2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 120402001, Environmental Policy Bureau, Ministry of the Environment, Japan)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidance Document, No.23, September 2000
Version / remarks:
OECD Guidance Document, No.23, September 2000, "Guidance Document on Aquatic Toxicity
Testing of Difficult Substances and Mixtures"
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Chemical name: (Z)-1-chloro-2,3,3,3-tetrafluoropropene
Another name: HCF0-1224yd(Z)
Purity of test item: >99%(GC area percentage)
Supplier: ASAHI GLASS CO., LID.
Lot number: 151007
The test item was treated as 100% in purity.
Analytical monitoring:
yes
Details on sampling:
Concentration of test item in test solution
Frequency of measurement: At the start of the exposure, 24 and 48 hours after the start of exposure, and the end of exposure.
Sample for measurement: Another solution sampled from the preparation container (at the start of exposure). The test solution taken out from the test vessels in each test level for analytical chemistry ( at 24 and 48 hours after the start of the exposure). The mixed solution taken out with equal volume of the test solution from the test vessels in each test level (at the end of exposure)
Volume of sample: Approximately 10-100 mL (all test levels)
Vehicle:
no
Details on test solutions:
Culture medium
At the pre-culture and algal growth inhibition test, the OECD medium (OECD TG 201; March 23, 2006) prepared with purified water was used.


Preparation of test solution
After medium was added in sampling bag for gas analysis, gaseous test sample was enclosed in the bag. The bag was shaken about 2 hours. After that, the solution was settled for more than 12 hours about 22°C to produce the stock solution. At the day that the test solution was prepared, test item concentration of stock solution was measured. Required volume of stock solution and medium were mixed in preparation container, and stirred to prepare the test solution. The test solution was divided into each test vessel.

Volume of test solution
600 mL/test level (100 mL/test vessel) (One additional vessel for analytical chemistry of the test item at 24 and 48 hours was set, respectively.)
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism
Species: Pseudokirchneriella subcapitata
Reason for selection of species: Species recommended in the test guidelines
Source: American Type Culture Collection
Strain number: ATCC22662
Supplied date: June 30, 1995
Subculture: Passage cultured under sterile conditions in this laboratory
Confirmation of reproducibility of test system: Algae growth inhibition test with a reference substance was periodically conducted. The latest data is shown below.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21-24 °C (not varied more than ±2°C)
Nominal and measured concentrations:
Nominal: 0 and 200 mg/l
Measured: 0 and 4.87 mg/l
Details on test conditions:
Test apparatus and equipment
Test vessel: Sterilized 500 mL Erlenmeyer flask ( closed vessel)
Incubator: Incubator with temperature and illumination control, continuous shaking (Incubator with rotary shaker and artificial illumination, U.S.1 Corp.)

Test conditions
Type of test: Incubation with rotary shaking (approximately 100 rpm), closed system
Duration: 72 hours
Test concentration: Approximately 200 mg/L (nominal) as limit test. The test concentration was decided based on the results of preliminary study.
Control: The medium without the test item

Replicate: 6 replicates/test level (One additional replicate for analytical chemistry of the test item at 24 and 48 hours was set, respectively.)
Volume of test solution: 600 mL/test level (100 mL/test vessel) (One additional vessel for analytical chemistry of the test item at 24 and 48 hours was set, respectively.)
Initial cell concentration: The algae were counted in pre-culture incubated under the same conditions as the test for 3 days (from December 12 to December 15, 2015) as inoculums, and were added to test vessels to bring the initial cell concentration of 0.50x10^4 cells/mL.
Operation: All operations were carried out under sterile conditions.
Temperature: 21-24 °C (not varied more than ±2°C)
Light intensity: Nominal 90 µmol·m^-2s^-1 ±20% (within±15% from the average light intensity) Continuous illumination provided by fluorescent lights with wavelength range of 400-700 nm

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 4.87 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.:
>= 4.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Observation and measurement of test solution
a) Appearance of test solution: At the start of exposure, test solutions of all test levels were colourless and clear. At the end of exposure, the appearance of test solution of all test levels were green due to the algal growth.
b) Water quality and environmental conditions: The measured values of pH were 7.8-9.7. The pH of control varied more than the range described in the guideline (not varied more than 1.5 in usual case). Culture temperatures in incubator were 22.6-23.20 °C and light intensities were 88-90 umol m^-2 s^-1.
c) Concentration of test item in test solution: The measured concentration of the test item in the test solutions were 152 mg/L at the start of the exposure, 2.84 mg!L at 24 hours after the start of exposure (1.87% of the concentration at the start), 2.02 mg/Lat 48 hours after the start of exposure (1.33% of the concentration at the start) and 2.66 mg/L at the end of exposure (1.75% of the concentration at the start). The concentration of the test item in the test solution was 4.87 mg/L by geometric mean of the measured concentration.

ErC50 of the test item based on the growth rate was >4.87 mg/L.

The algal growth in exposure level was same as the control. As the results of cell observation, the condition of cells in the control was not abnormal and that in exposure level was same as the control. On the growth rate, there was not statistical difference in exposure level. By the results in statistical analysis and cell observation showed above, NOEC based on growth rate was estimated at >=4.87 mg/L.

Validation criteria
a) Growth of the control: The cell in the control grew exponentially during the exposure. At the end of exposure, it increased to 74 or more times of the number of initial cells in the control.
This meets the validity of the test: the cell growth in control should have increased by a factor of at least 16 times at 72 hours after the start of the exposure.
b) The specific growth rates of section-by-section in the controls: The mean coefficient of variation for section-by-section specific growth rate in the controls was 29%. It meets the validity of the test: the mean coefficient of variation in the control must not exceed 35%.
c) The specific growth rates in replicate controls: The coefficient of variation of specific growth rate in replicate controls was 1.4%. It meets the validity of the test: the coefficient of variation in controls must not exceed 7%.

Discussion
This study was conducted as a limit test in order to confirm the effect on the test organisms around upper limit concentration in test methods in medium. It was difficult to prepare the test solution of nominal concentration because the test item is gaseous substance. Accordingly, definitive study was carried out at approximately 200 mg/L (nominal) level in expectation of loss of the test item in test solution at experimental operation. As the results, in 152 mg/L at the start (higher than 100 mg/L of upper limit concentration in test methods), there was no adverse effect on the test organisms. Therefore, the test item had no adverse effect on the test organisms at 100 mg/L of upper limit concentration in test methods. The concentrations of the test item in the test solutions were significantly decreased during the exposure. The reason was estimated to the limit in algae growth inhibition study of the volatile substance (volatilization to headspace in the test vessel). With regard to the environmental conditions of the test, the excess increase of pH in control, which is out of the range of the regulation of the test method, was observed. It was decided that the excess increase of pH in control was due to the limit of the algae growth inhibition study with volatile substance (gas exchange between outside and inside of the test vessel is impossible because of the test vessel of closed system). The environmental conditions without pH were within the suitable range. Therefore, it is concluded that this study complied with the applied test guidelines.
Results with reference substance (positive control):
Reference substance: Potassium dichromate (JIS special grade, W ako Pure Chemical Industries, Ltd. Lot No. RCK7838)
Test period: November 24 to November 27, 2015
ErC50 (0-3d); 1.1 mg/L
This value was within the normal range of the reference substance in this laboratory (mean± 2S.D.) [mean± S.D.: 1.0 ± 0.19 mg/L (n=28)].

pH of test solutions

Measured concentration (mg/l)a

pH at start

pH at end

Control

7.8

9.7

4.87

7.8

9.6

a: Geometric mean of the measured concentrations (also expressed as measured concentration in the following tables and figure).

Culture temperature ad light intensity in incubator

Time

Start

1-day

2-day

End

Culture temperature (°C)

22.7

22.6

22.7

23.2

Light intensity (umol m-2s-1

88

90

88

89

 

Value of biomass at each time

Measured concentration (mg/l)a

No.

Cell concentration ( x 104cells/mL)

0 hourb

24 hours

48 hours

72 hours

Control

A

0.5

3.1

15

43

B

0.5

3.0

14

37c

C

0.5

3.2

15

41

D

0.5

3.1

15

38

E

0.5

3.4

14

41

F

0.5

3.1

14

39

Mean

0.5

3.1

14

40

Standard Deviation

0

0.14

0.57

2.4

4.87

A

0.5

3.5

14

41

B

0.5

3.2

13

40

C

0.5

3.2

13

39

D

0.5

3.3

13

39

E

0.5

3.3

14

40

F

0.5

3.3

14

39

Mean

0.5

3.3

13

40

Standard Deviation

0

0.12

0.44

0.92

a: Geometric mean of the measured concentrations (also expressed as measured concentration in the following tables and figure).

b: The value based on the measured value of pre-culture

c: The minimum cell growth in control (biomass at the end of exposure I biomass at the start of exposure) - 37 /0.5 =74

 

 

 

Growth rate and growth inhibition

Measured concentration (mg/l)a

No.

Growth rate (0-3 day)

Inhibition rate (%)

Control

A

1.49

 

B

1.43

 

C

1.47

 

D

1.44

 

E

1.47

 

F

1.45

 

Mean

1.46

 

Standard Deviation

0.0203

 

4.87

A

1.47

-0.70

B

1.46

0.18

C

1.45

0.49

D

1.45

0.53

E

1.46

-0.087

F

1.45

0.74

Mean

1.46

0.19

Standard Deviation

0.00767

0.53

 

ErC50 and NOEC

ErC50 (mg/l)

NOEC (mg/l)

>4.87

≥4.87

 

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of HCFO 1224yd(Z) to algae was measured in a 72 -hour study conducted in accordance with OECD TG 201. The EC50 (growth rate) was reported to be >4.87 mg/l and the NOEC (growth rate was reported to be >=4.87.
Executive summary:

The toxicity of HCFO 1224yd(Z) to algae was measured in a 72 -hour study conducted in accordance with OECD TG 201. The EC50 (growth rate) was reported to be >4.87 mg/l and the NOEC (growth rate was reported to be >=4.87.

Description of key information

The toxicity of HCFO 1224yd(Z) to algae was measured in a 72 -hour study conducted in accordance with OECD TG 201. The EC50 (growth rate) was reported to be >4.87 mg/l and the NOEC (growth rate was reported to be >=4.87.

Key value for chemical safety assessment

EC50 for freshwater algae:
4.87 mg/L
EC10 or NOEC for freshwater algae:
4.87 mg/L

Additional information