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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 - 16 Nov 2006
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 0.95, 3.1, 9.8, 31 and 100 mg/L
- Sampling method: Duplicate samples were taken at the beginning and the end of the test.
- Sample storage conditions before analysis: Samples were stored in the freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest test concentration of nominal 100 mg/L was prepared by dissolving 25 mg test item into 250 mL culture medium by intense stirring for 15 min. Adequate volumes of this test medium were diluted with culture medium to prepare the test media.
- Differential loading: no
- Controls: yes, test medium control
- Eluate: no
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no, test solutions were clear and colorless throughout the study period.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Obtained from “The Collection of Algal Cultures” of the Institute for Plant Physiology, University Goettingen, Germany.
- Age of inoculum (at test initiation): 3
- Method of cultivation: The algae are cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

ACCLIMATION
- Culturing media and conditions (same as test or not):The cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 ± 2 °C
pH:
7.9 at test start
8.1 - 8.4 at test end
Nominal and measured concentrations:
nominal: control, 0.95, 3.1, 9.8, 31, 100 mg/L
measured: < LOQ, 85% of nominal, 91% of nominal, 99% of nominal, 96% of nominal, 95% of nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks covered with glass dishes
- Material, size, headspace, fill volume: glass, 50 mL, headspace: 20 mL, fill volume: 30 mL
- Aeration: no, but constantly shaken
- Initial cells density: 5000 cells/mL
- Control end cells density: 724000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD guideline 201

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination in the incubator (24 h/day)
- Light intensity and quality: 7320-7800 lux (minimum and maximum value of measurements at 6 places distributed over the experimental area at the surface of the test media)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometrical measurement after 24, 48 and 72 hours
- Microscopic determination: In the test concentration of 100 mg/L the shape of algal cells was examined compared to the control.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 3.2
- Range finding study: yes, but no further information are given
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
9.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30.62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 29.319 - 31.981 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13.37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 11.637 - 14.882 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 14.621 - 14.815 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 7.489 - 7.624 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 17.001 - 17.367 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 7.683 - 7.963 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Unusual cell shape: At the microscopic examination of the shape of the algal cells after 72 h test period no difference was observed between the algae growing in the test concentration of nominal 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells growing at least up to this test concentration was obviously not affected.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, all test solutions were clear and colorless.
Results with reference substance (positive control):
Results not reported.
Reported statistics and error estimates:
Based on the calculated cell densities, the ErC50, EbC50, and EyC50 and the corresponding EC10 values and their 95%-confidence limits were calculated by Probit analysis. For the determination of the LOEC and NOEC, the calculated growth rates, areas under the growth curve and yields at the test concentrations were tested on significant differences to the control values by the Bonferroni t-test (growth rate, area under the growth curve) and Williams test (yield), respectively (ToxRat Version 2.09, 2001-2005).

Table 1: Cell density at different test substance concentrations during test (average* values)

Nominal concentration (mg/L)

Average Cell Density
(initial cell density: 5,000 cells/mL)

After 24 h

After 48 h

After 72 h

Control

35000

149000

724000

0.95

36000

145000

737000

3.1

31000

141000

743000

9.8

28000

138000

567000

31

24000

49000

60000

100

-5000

-8000

6000

* Number of samples: control: 6 flasks; each test substance concentration level: 3 vessels

Table 2: Yield y and percentage inhibition of y during the test period and results of the Williams Test (α = 0.05, one-sided) with the yield y

Nominal concentration (mg/L)

Yield y [mg/L] and % inhibition of y

0-24 h

0-48 h

0-72 h

y

%

t-values

y

%

t-values

y

%

t-values

Control

3.04

0.0

-

14.41

0.0

-

71.88

0.0

-

0.95

3.13

-2.9

0.599-

13.99

2.9

-1.325-

73.23

-1.9

1.709-

3.1

2.60

14.5

-2.994+

13.60

5.6

-2.540+

73.76

-2.6

1.709-

9.8

2.32

23.7

-4.910+

13.32

7.6

-3.423+

56.25

21.7

-16.510+

31

1.90

37.6

-7.783+

4.36

69.8

-31.583+

5.48

92.4

-701.144+

100

-0.44

114.5

-23.691+

-0.50

103.5

-46.837+

0.14

99.8

-75.790+

Table 3: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

145 fold

yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

16.1

Yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

1.6%

Yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
NOErC (72 h) = 9.8 mg/L
ErC50 (72 h) = 30.62 mg/L
ErC10 (72 h) = 13.37 mg/L
Executive summary:

The influence of p-chloro-m-cresol on the growth of the green alga subspicatus (formerly Scenedesmus subspicatus) was investigated by Vinken & Wydra (2007) in a 72 hours static test according to the following guidelines: OECD 201 (2006), Directive 92/69/EEC, C.3 (1992). The calculated average growth rates decreased in a dose dependent manner. The 72 h NOEC value was 9.8 mg a.i./L, based on the lack of a statistical growth inhibition at these concentrations. Based on the mean area under the growth curve, the 72-hour EbC50 value was 17.2 mg a.i./L. Based on growth rate, the 72-hour ErC50 and the Er10 values were 30.6 mg a.i./L and 13.37 mg a.i./L, respectively. Mean measured concentrations ranged from 97 % at test start to 89 % at test end of the nominal concentration during the test. Therefore, all endpoints were based on nominal concentrations of the test item.

Validity criteria for the acceptance of the test can be considered as fulfilled.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to analogue justification document provided in IUCLID section 13.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
9.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30.62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
95% CL: 29.319 - 31.981 mg/L
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13.37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
95% CL: 7.489 - 7.624 mg/L
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
yield
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks:
95% CL: 14.621 - 14.815 mg/L
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks:
95% CL: 7.489 - 7.624 mg/L
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
95% CL: 17.001 - 17.367 mg/L
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
95% CL: 7.683 - 7.963 mg/L
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel. 1, Vinken & Wydra 2007

Description of key information

NOErC (72 h) = 9.8 mg/L (nominal, Desmodesmus subspicatus), read-across

ErC50 (72 h) = 30.62 mg/L (nominal, Desmodesmus subspicatus), read-across

Key value for chemical safety assessment

EC50 for freshwater algae:
30.62 mg/L
EC10 or NOEC for freshwater algae:
9.8 mg/L

Additional information

No study investigating the toxicity of sodium p-chloro-m-cresolate (CAS 15733-22-9) to algae is available. Therefore, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across to the source substance (p-chloro-m-cresol, CAS 59-50-7) which isthe main transformation product of the target substancesodium p-chloro-m-cresolate (CAS 15733-22-9) is applied.

The source substancep-chloro-m-cresol is the common compound in this analogue approach and is solely responsible for the (absence of) effects.Thus, the source substance is considered a suitable representative for the evaluation of the toxicity of the target substance to aquatic organisms. The read-across approach is justified in detail within the analogue justification in IUCLID section 13.

One key (Vinken & Wydra 2007) is available investigating the toxicity of p-chloro-m-cresol (CAS 59-50-7) to aquatic algae. The key study was conducted according to OECD 201 (2006) and Directive 92/69/EEC, C.3 (1992). Algae were exposed to the test item for 72 hours under static conditions to nominal concentrations of 0, 0.95, 3.1, 9.8, 3.1, 9.8, 31, 100 mg/L. Cell concentrations were measured after 24, 48 and 72 hours. The calculated average growth rates decreased in a dose dependent manner. The 72-hour NOEC value was 9.8 mg/L, based on the lack of a statistical growth inhibition at these concentrations. Based on the mean area under the growth curve, the 72-hour EbC50 value was 17.2 mg/L. Based on growth rate, the 72-hour ErC50 value was 30.6 mg/L. Mean measured concentrations ranged from 97 % at test start to 89 % at test end of the nominal concentration during the test. Therefore, all endpoints were based on nominal concentrations of the test item.

Based on the available results from a structurally similar source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which is the main transformation product of the target substance and is characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that sodium p-chloro-m-cresolate has effects in the same range as the target substance.