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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:
Exposure period: Duration of 3 days; 8-11 May 2001, 08-May-2001 to 06-Sep-2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid and conclusive guideline study under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
- To provide the algae with sufficient inorganic carbon, an extra amount of NaHCO3 was added to the algal medium. The NaHCO3 was 250 mg/L, while the normal NaHCO3 concentration was 50 mg/L in algal medium.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: On day 0 and 3 samples of 0.5 mLl were taken from all test solutions. On day 1 and 2 samples were taken from the test solutions with test substance only.
- Sampling method: On day 0 and 2 samples of 0.5 mL were taken in duplicate (one sample served as contra sample and was stored at a temperature of 2 - 8°C) from all test flasks.
- Sample storage conditions before analysis: refrigerated (2 - 8 °C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- On day 0 a stock solution with TCP concentration of 160 mg/L was prepared by dissolving 115 µL (160 mg) TCP in 1 litre algal medium. Per concentration 3 sterile serum bottles of 130 mL were filled (see dilution for details). The bottles were closed with a screw cap with teflon inlay. Six control bottles were prepared. Before inoculation of test solutions with algae, the test bottles were placed for one hour in the thermostated incubator shaker with a temperature of 23°C.
- Dilution: 0, 8.0, 16, 33, 65 or 130 mL of stock solution were adjusted each with the respective volume of alga medium (table 1) to 130 mL to give final solutions of 0, 10, 20, 40, 80 and 160 mg/L of test item.
- Chemical name of vehicle: no vehicle
- Evidence of undissolved material: none reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: freshwater algae
- Strain: ATCC 22662
- Source: Culture Collection of Algae and Protozoa, Ambleside, Cumbria, UK (delivery date: 6 October 2000)
- Age of inoculum: Erlenmeyer flasks with medium were inoculated with the algae an 9 October 2000
- Method of cultivation: The algae are cultured in 100 mL sterile Erlenmeyer flasks with 50 mL sterilised medium. Each week the culture is transferred into fresh algal medium containing 50 mg/L NaHCO3 (for composition see Table 1). The preculture used for this study was transferred 4 days prior to the start of the test. The temperature setpoint of the culture chamber is 23°C. The culture chamber illuminated 24 hours a day with fluorescent lamps.


ACCLIMATION
- Acclimation period: see above
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
Not explicitly stated in the report, but may be derived from the test media composition, see table 1 in “any other information on materials and methods including tables”
Test temperature:
22.4 - 24 °C
pH:
8.0 - 9.8, see table 2 in “any other information on materials and methods including tables” for details
Dissolved oxygen:
not applicable
Salinity:
Not explicitly stated in the report, but may be derived from the test media composition, see table 1 in “any other information on materials and methods including tables”
Nominal and measured concentrations:
Nominal: 0, 10, 20, 40, 80, 160 mg/L
Measured: 0, 7.3, 12.8, 24.8, 48.3 and 101 mg/L
See table 3 in “any other information on materials and methods including tables” for details
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: sterile serum bottle , 130 mL of volume, completely filled, no headspace
- Aeration: no
- Initial cells density: 10'000 cells/mL
- Control end cells density: 1'525'000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes, see table 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see table 1
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: On day 0 the light intensity at the photosyntheticalty effective wavelength range of 400 nm to 700 nm was measured in the shaking incubator at the level of the serum bottles with the Li-Cor 0047-005. The light intensity at the level of the algae should be 60 - 120 µE/m².s (ISO, 1989: ISO 8692: Water quality - Fresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricornutum. International Organization for Standardization, Geneva, Switzerland)


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: counting chamber and spectrophotometer
- Chlorophyll measurement: To determine the effect of 1,2,3-trichloropropane on growth of the algae, the absorption of the test solutions at 750 nm was measured during the test. The absorption was measured 24, 48 and 72 hours alter the start of the test in all serum bottles using a spectrophotometer (Hitachi U-2000) and a cuvette with a pathlength of 5 cm. The volume, used for measurement of the absorption (10 mL), was not returned to the serum bottles.
- the biomass integral was calculated as laid down in the ISO guideline ISO 8692: Water quality - Fresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricornutum International Organization for Standardization, Geneva, Switzerland, 1989. The element basis (i.e. number of cells/mL, area under the curve, growth rate, etc.) used was area under the curve.
- the growth rate was calculated: µ = ln(N3/N0)/3 (where Nn is the cell density at day n in cells/mL)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes, but not reported

Reference substance (positive control):
yes
Remarks:
- historical control with potassium dichromate (for details see below)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
49.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
101 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
and growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: no
The difference between the nominal and mean measured concentrations was more than 20 % and for this reason the endpoints of the test were based on mean measured concentrations.
Results with reference substance (positive control):
- Historical positive control showed sensitivity, see above in section “any other information on materials and methods including tables” for details
Reported statistics and error estimates:
- The No Observed Effect Concentration (NOEC), based on biomass integral at test termination, was determined with Williams' Test (Williams DA 1972). The comparison of several dose levels with a zero dose control Biometrics 27: 103-117.)
- Determination of EC50: Using linear interpolation, with mean measured concentrations on a logarithmic scale (base = 10).

Table 4: Mean measured algal cell densities during the test periodA

Measured concentration
(mg/L)

Mean cell density x 104(cells/mL)

Day 1

Day 2

Day 3

0

2.716

31.55

152.5

7.3

2.475

28.93

142.3

12.8

2.533

28.78

139.7

24.8

1.991

24.50

109.5

48.3

1.121

18.43

76.38

101

1.237

8.831

30.05

On day 0 the cell density of the preculture was determined. Based on this result a quantity 0.265 mL of the preculture was added to all serum bottles resulting in an initial concentration of 1.0 x 10 exp 4 cells/mL.

The measured algal cell densities given in table 5 are mean cell densities of all serum bottles. The data show that the cell density increased 134 — 174 times in the controls, which meets the validity criterion of a minimal 16 times increase.

Table 5: Mean biomass integral and biomass integral inhibition (%) on day 3

Measured concentration
(mg/L)

Biomass integral x 104

Biomass integral inhibition
on day 3 (%)

Day 1

Day 2

Day 3

0

0.86

17.0

108

0

7.3

0.74

14.4

100

7.41

12.8

0.77

15.4

98.7

8.66

24.8

0.50

12.7

78.7

27.1

48.3

0.06

8.83

55.2

48.9

101

0.12

4.15

22.6

79.1

The cell densities (unrounded data) of each serum bottle were used to calculate the area under the growth curve (biomass integral). The mean biomass Integral and the biomass Integral inhibition is given in Table 4 for each test concentration.

Table 6: Mean growth rate inhibition (%) at various measured concentrations

Measured concentration
(mg/L)

Growth rate inhibition (%)

Day 0-1

Day 1 -2

Day 2 - 3

Day 0 - 3

7.3

9.6

-0.4

-1.2

1.3

12.8

6.9

1.1

-0.4

1.8

24.8

32

-2.6

4.9

6.5

48.3

99

-18

9.7

14

101

85

18

22

32

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of 1,2,3-trichloropropane against aquatic algae was tested in a 72h static test according to OECD TG 201:
EC50-72h = 49.6 mg/L based on measured concentrations and biomass integral inhibition as effect parameter
EC50-72h =101 mg/L based on measured concentrations and growth rate inhibition as effect parameter
NOEC-72h = 12.8 mg/L based on measured concentrations and biomass integral inhibition as effect parameter
Executive summary:

The toxicity of 1,2,3-trichloropropane to algae (Pseudokirchnerella subcapitata) was tested according to OECD GLP Principles of Good Laboratory Practice and Compliance Monitoring No. 1, ENV/MC/CHEM(98)17 of 21 January 1998 and according to OECD Guideline 201 (OECD 1984) with an adaptation of the algal medium (extra amount of NaHCO3) to enable the use of a closed system. 1,2,3-Trichloropropane is volatile and for this reason algae were exposed for three days using closed system flasks containing 130 mL test solution. 1,2,3-Trichloropropane was tested at nominal test concentrations of 0, 10, 20, 40, 80 and 160 mg/L in algal medium.

To study the exposure of the algae to 1,2,3-trichloropropane, samples of the test solutions were taken at the start, day 1, day 2 and on day 3 of the test and analyzed with Gas Chromatography. The difference between the nominal and the mean measured concentrations was larger than 20 % and for this reason the biological endpoints of the test are based on the average of the measured concentration being 0, 7.3, 12.8, 24.8, 48.3 and 101 mg/L.

The cell density increased 134 — 174 times in the controls, which meets the validity criterion of a minimal 16 times increase. The cell density measurements were used to calculate the biomass integral and growth rate for each serum bottle. Using linear interpolation, with mean measured concentrations on a logarithmic scale (base = 10), the EC50 0-72h for biomass integral and growth rate were 49.6 and > 101 mg/L, respectively. The No Observed Effect Concentration (NOEC), based on biomass integral at test termination, was determined with Williams' Test (Williams 1972). Based on mean measured concentrations this revealed a NOEC of 12.8 mg/L.

Description of key information

The acute toxic effects of 1,2,3 TCP to algae are characterized by a EC50 of 49.6 mg/L and a NOEC of 12.8.

Key value for chemical safety assessment

EC50 for freshwater algae:
101 mg/L
EC10 or NOEC for freshwater algae:
12.8 mg/L

Additional information

Data exist from three algal species (Chlorella vulgaris, Chlamydomonas angulosa, Pseudokirchnerella subcapitata). Hutchinson et al (1980) tested the former two species in 3 h exposure experiment were inhibition of metabolic rate was measured as reduction of carbon dioxide uptake. The determined EC50 values of 170 and 112 mg/L respectively were not regarded for assessment as the method is considered not acceptable.

Reliable figures are available form de Groot (2002) on green alga (Pseudokirchnerella subcapitata). The study is valid and conclusive and a 72-h ErC50 value of 101 mg/L (i.e. related to the endpoint growth rate), which is the relevant value with regard to GHS/CLP, was obtained. This study (de Groot 2002) finds an acute 72-h NOEC of 12.8 mg/L.