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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 2017-11-29 to 2017-12-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
March 23, 2006,
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Lot number: 707001
Purity: 98.6%
Appearance: Clear and colourless liquid
Storage conditions: Cold and dark storage place
Analytical monitoring:
yes
Details on sampling:
Samples were taken to measure the concentration of test item in test solution at the start of exposure, 24 and 48 hours after the start of exposure and at the end of exposure. Sample volumes were approximately 10 mL (at the start of exposure), 100 mL (at 24 and 48 hours after the start of exposure) and approximately 12 mL (at the end of exposure) for all test levels.
Vehicle:
no
Details on test solutions:
A weighed test sample of 230 mg and culture medium of 2300 mL were mixed to prepare the nominal concentration of 100 mg/L and then gently stirred with a magnetic stirrer for 48 hours. The solution was then allowed to settle for 1 hour and the middle layer removed. The middle layer solution was collected as the stock solution. The test solution was prepared in a preparation container by mixing required volume of the stock solution and the medium processing similar to the control and divided into each test vessel. In the 100% test concentration level, the stock solution was used for the test solution.

Test solution appearance was observed at the start and end of exposure. Test solution pH was measured from a solution sampled separately from the preparation container at the start of exposure and from one test vessel in each test level at the end of exposure, using a pH meter (Model HM-21P (DKK-TOA CORPORATION)). Light intensity was measured at the start, 24 hours and 48 hours after the start, and at the end of exposure using a glass thermometer using a quantum scalar laboratory irradiance meter (Model LI-250 (LI-COR, Inc.). Culture temperature was measured at the start, 24 hours and 48 hours after the start, and at the end of exposure using a glass thermometer.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source: American Type Culture Collection
Strain number: ATCC 22662
Supplied date: June 30, 1995
Subculture: Cultured under sterile conditions in the testing laboratory
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None
Post exposure observation period:
NA
Hardness:
Not specified
Test temperature:
21 - 24°C
pH:
7.8 - 9.5
Dissolved oxygen:
Not specified
Salinity:
NA
Conductivity:
Not specified
Nominal and measured concentrations:
Measured concentrations (geometric mean): Control, 0.0165, 0.0184, 0.0428, 0.0874 and 0.217 mg/L
Details on test conditions:
Type of test: Incubation with rotary shaking (approximately 100 rpm) closed system.

Replicates: 6 replicates/control
3 replicates/exposure level
(two additional replicates for analytical chemistry of the test item were set for 24 and 48 hours)

Volume of test solution: 600 mL/control (100 mL/test vessel)
300 mL/exposure level (100 mL/test vessel)
(two additional replicates for analytical chemistry of the test item were set for 24 and 48 hours)

Light intensity: Nominal 90 µmol·m-2·s-1 in wavelength range of 400-700 nm within ±20% of nominal (within ±15% from the average light intensity). Continuous illumination provided by alternative LED light to fluorescent lamp. Light intensities were 87-91 µmol·m-2·s-1 throughout the study.

Initial cell concentration: Algae were counted in pre-culture incubated under the same conditions as the test for 3 days (from December 1 to December 4, 2017) as inoculums, and were added to test vessels to bring the initial cell concentration of 0.50 E+04 cells/mL

All operations were carried out under sterile conditions.

Observation and measurements:
Algal biomass (cell concentration) was measured every 24 hours after the start of exposure (the blank correction was conducted by measuring the value of blank solution of test vessel prepared for background). Cell condition was observed in one vessel in each test level at the end of exposure. Observations were made using a particle counter (Model COULTER Z2 (Beckman Coulter, Inc., Instrument No. CC-004)) and system biological microscope (Model BX41 (Olympus Corporation)).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.217 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.087 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The algal growth in 0.217 mg/L exposures was logarithmic, although a little growth inhibition was reported. Growth inhibition in the 0.0874 to 0.0165 mg/L exposures were the same as the control. Algal cell conditions in all exposure levels were the same as the control in that there were no abnormalities.

Statistical differences in growth rate were seen in 0.217 and 0.0184 mg/L levels. The mean growth inhibition rate in the 0.0184 mg/L level was 2.3%. Although there was a significant difference in this exposure level, it was judged that the test item caused no adverse effect on the test organism. The average inhibition rate at 0.0184 mg/L level was low and there was no obvious correlation in concentration-response of the growth inhibition rate of each test vessel in 0.0874 to 0.0165 mg/L levels. From the statistical analysis of the test results, a growth rate based NOEC of 0.0874 mg/L was estimated.

All validity criteria prescribed by guideline were met. Cell growth was exponential in the control during the exposure, having increased to a factor of 64 at the end of exposure (i.e. greater than a factor of 16). The mean coefficient of variation for section-by-section growth rates in the control cultures was 6.0% (i.e. <35%). The coefficient of variation of specific growth rates in replicate control cultures was 0.56% (i.e. <7%).
Results with reference substance (positive control):
Periodically, an algae growth inhibition test of a reference substance (potassium dichromate) using the test organisms is conducted to confirm the reproducibility of the test system, the most recent of which (as of the date of the current study) showed results (72-h ErC50 = 1.1 mg/L) within the stipulated range to background data in the testing laboratory.
Reported statistics and error estimates:
The mean value of biomass for each test level was plotted against time to produce growth curves. Using this curve, inhibition rates were calculated comparing with control values on growth rate. The specific growth rate for a specific period was calculated as the logarithmic increase in biomass.

Specific growth rate over the exposure duration was calculated for determination of ErC50 and NOEC. In the control, specific growth rates for section-by-section were calculated to check test validity.

The percentage inhibition for each exposure level was mean value of the percent inhibition in average specific growth rate for a replicate (Iµ) in test level. The percent inhibition (Iµ) was calculated from mean value for average specific growth rate in control (µc), average specific growth rate for each replicate in exposure level (µT) via the following formula:

Iµ = ((µc - µT) / µc) x 100

ErC50 estimation: Since no less than 50% growth inhibition was obtained within the exposure levels, the ErC50 was estimated as “> the highest test concentration”.
NOEC estimation: Bartlett’s test was used to determine the homogeneity of variance for the data. Then one-way analysis of variance and Dunnett’s multiple comparison test was used to determine the significant difference between the control and the exposure levels. The statistical analysis was conducted using Microsoft Excel. The NOEC was determined by the results of statistical analysis and the total test results.

Value of biomass at each time

 

Measured concentration

(mg/L)

No.

Cell concentration (1E+04 cells/mL)

0 houra

24 hours

48 hours

72 hours

Control

A

0.50

1.9

8.4

33

B

0.50

2.0

8.9

32b

C

0.50

1.9

8.9

32b

D

0.50

2.0

8.6

32b

E

0.50

2.1

8.7

34

F

0.50

1.9

8.7

33

Mean

0.50

2.0

8.7

33

S.D.

0

0.071

0.17

0.76

0.0165

A

0.50

2.1

9.0

31

B

0.50

2.0

8.8

32

C

0.50

2.0

8.6

30

Mean

0.50

2.0

8.8

31

S.D.

0

0.063

0.21

1.1

0.0184

A

0.50

1.9

8.6

29

B

0.50

2.1

8.5

30

C

0.50

1.9

8.9

30

Mean

0.50

1.9

8.7

30

S.D.

0

0.11

0.17

0.77

0.0428

A

0.50

1.9

8.5

30

B

0.50

2.0

8.8

32

C

0.50

2.1

9.0

30

Mean

0.50

2.0

8.8

30

S.D.

0

0.078

0.25

1.0

0.0874

A

0.50

2.0

9.2

30

B

0.50

2.0

9.1

32

C

0.50

2.0

9.2

30

Mean

0.50

2.0

9.2

31

S.D.

0

0.019

0.011

1.3

0.217

A

0.50

2.0

8.3

24

B

0.50

2.0

7.9

29

C

0.50

1.8

8.5

27

Mean

0.50

2.0

8.2

27

S.D.

0

0.11

0.31

2.1

aThe value based on the measured value of pre-culture

bThe minimum cell growth in control (biomass at the end of exposure / biomass at the start of exposure) 32/0.5 = 64

Growth rate and growth inhibition rate

 

Measured concentration

(mg/L)

No.

Growth rate

(0-3 d)

Growth inhibition rate (%)

Control

A

1.40

-

 

B

1.39

-

 

C

1.38

-

 

D

1.38

-

 

E

1.40

-

 

F

1.39

-

 

Mean

1.39

-

 

S.D.

0.00774

-

 

0.0165

A

1.38

0.80

 

B

1.39

0.35

 

C

1.36

1.9

 

Mean

1.38

1.0

 

S.D.

0.0113

0.81

 

0.0184

A

1.35

3.0

 

B

1.37

1.8

 

C

1.36

2.0

 

Mean

1.36

2.3

 

S.D.

0.00878

0.63

 

0.0428

A

1.36

1.9

 

B

1.38

0.64

 

C

1.36

2.1

 

Mean

1.37

1.6

 

S.D.

0.0111

0.80

 

0.0874

A

1.37

1.8

 

B

1.39

0.085

 

C

1.37

1.7

 

Mean

1.37

1.2

 

S.D.

0.0136

0.98

 

0.217

A

1.30

6.8

 

B

1.35

3.1

 

C

1.33

4.2

 

Mean

1.33

4.7

 

S.D.

0.0267

1.9

 

 

Measured concentrations of test item in test solutions

 

Stock solution content

(%)

Measured concentration (mg/L)

(Percentage of measured concentration versus that at the start %)

At the start

24 hours

48 hours

At the end

Geometric mean

Control

n.d.

n.d.

n.d.

n.d.

-

6.25

0.205

n.d.a

n.d.a

n.d.a

0.0165

12.5

0.389

n.d.a

n.d.a

n.d.a

0.0184

25.0

1.00

0.0243

(2.43)

0.0225

(2.25)

0.0206

(2.06)

0.0428

50.0

1.89

0.0528

(2.76)

0.0418

(2.21)

0.0485

(2.56)

0.0874

100

6.37

0.110

(1.73)

0.113

 (1.77)

0.108

(1.70)

0.217

n.d.: <0.02 mg/L

a: In accordance with OECD Guideline Document No. 23, half the determination limit of the test item (0.01 mg/L) was adopted for the calculation of geometric mean, because the test item was detected but not quantified.

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour ErC50 and NOEC (growth rate) values were determined to be >0.217 and 0.0874 mg/L, respectively.
Executive summary:

A study investigating the toxicity of the test item to the freshwater algae, Pseudokirchneriella subcapitata, exposed to the test item for 72-hours was assessed according to the OECD 201 ‘freshwater alga and cyanobacteria, growth inhibition test’ Guideline.

Tests were conducted within the substance’s limit of solubility and there were no factors which may have affected the reliability of the test. Algal growth inhibition was limited, with 50% inhibition not occurring during the test in any of the exposure concentrations, therefore the 72-hour ErC50 was estimated to be >0.217 mg/L and the 72-h NOEC was estimated to be 0.0874 mg/L.

Description of key information

A study investigating the toxicity of the test item to the freshwater algae,Pseudokirchneriella subcapitata, exposed to the test item for 72-hours was assessed according to the OECD 201 ‘freshwater alga and cyanobacteria, growth inhibition test’ Guideline. The study was conducted at a GLP accredited laboratory and met the validity criteria prescribed in the test guideline and is therefore considered to be valid and reliable.

 

Tests were conducted within the substance’s limit of solubility and there were no factors which may have affected the reliability of the test. Algal growth inhibition was limited, with 50% inhibition not occurring during the test in any of the exposure concentrations, therefore the 72-hour ErC50 was estimated to be >0.217 mg/L and the 72-h NOEC was estimated to be 0.0874 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.217 mg/L
EC10 or NOEC for freshwater algae:
0.087 mg/L

Additional information

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