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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:
2011-02-18 to 2011-02-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
For measurement of the actual concentration of the test substance, three samples (samples were taken out separately from replicate test beakers 4 to 6) were taken from the test medium of the single test concentration at the start of the test and at the end of the test using a Hamilton syringe (through septum). At the same sampling times, one sample was also taken from the solvent control (replicate 1).

The concentration of the test substance, 1,1,1,3,5,5,5-Heptamethyltrisiloxane, was analytically determined in each test medium sample immediately after sampling.

Twister extraction was performed which allows extraction of the test substance from the test water without removing the algae. Thus, samples had not to be filtered prior to analyses.
Vehicle:
yes
Details on test solutions:
Due to the low solubility of the test substance the organic solvent N,N-dimethylformamide (DMF) was used to solubilise the test item. The solvent was chosen based on its solubilising properties and its relative non-toxicity to algae.

The application solution for the dosage of the test concentration was prepared by dissolving 21.03 mg of the test item in 10 mL of DMF. Until application, the application solution was kept in a completely filled glass flask tightly closed with a septum lid.

For application, 13 µL of the application solution was injected with a Hamilton syringe into 130 mL of test water (through the septum from the closed glass flask). For the preparation of the solvent control, the same volume of DMF (without test item) was added to the test water.

The test medium was prepared just before the start of the test (i.e. exposure of algae).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Common name: The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum),

- Strain: 61.81 SAG

- Source: Supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).

- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure. 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.

- Reference substance: 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
24 mg/l as CaCO3
Test temperature:
21 °C
pH:
7.9 - 8.5
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control) and 0.20 mg/l.

At the start of the test, the measured test item concentration was 0.126 mg/l, corresponding to 63% of the nominal value. At the end of the test, the measured test item concentration was below LOQ (limit of quantification of the analytical method, LOQbio = 0.006 mg test item/l). The geometric mean measured test concentration was 0.019 mg/l, which corresponds to the solubility limit of the test item in water as determined in Harlan Laboratories Study B79727 (0.02 mg/l).

The biological results are related to the geometric mean measured test substance concentration and the mean initial measured test substance concentration.
Details on test conditions:
TEST DESIGN

The study design was based on the results of range-finding pre-tests (non-GLP).

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the algae at its solubility limit in test water. The single nominal concentration of 0.20 mg test item/l was tested. Additionally, a control (test water without test item) and a solvent control (100 µl DMF per litre) were tested in parallel.

The test design included six replicates of the single test concentration, three replicates of the control and six replicates of the solvent control.

The test was started using a nominal algal cell density of 10000 cells/ml. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM).

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


TEST VESSELS

Since the test item was determined to be volatile, the test was performed in 100-mL glass stoppered flasks with a septum completely filled with 130 mL of test medium, minimizing the air-space in the flasks and avoiding potential losses of test item. The test flasks were labelled with the study number and all necessary additional information to ensure unique identification.


TEST CONDITIONS

The test flasks were incubated in a temperature-controlled water bath at a temperature of 21 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7900 Lux (range: 7280 to 8420 Lux, measured at nine places in the experimental area). The light intensity over the incubation area deviated by less than ±15% from the average light intensity as recommended by the guideline.


DILUTION WATER

Reconstituted test water was prepared according to the test guidelines by dissolving analytical grade salts in sterile purified water. To keep the pH of the test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/l) were added to the test water.


OBSERVATIONS

In order to measure the algal biomass, a small volume of the algal suspension (250 µl at 24 and 48 hours, and 1000 µL at 72 hours) was withdrawn daily from each test flask (through septum) using a Hamilton Syringe. The volume was not replaced.

The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.

At the end of the test, a sample was taken from the solvent control and from the single test concentration to determine a potential influence of the test item on the shape and size of the algal cells by visual inspection.


WATER QUALITY

The pH was measured and recorded in each treatment at the start and end of the test. For the pH measurement at the start of the test, a separate test vessel was set up for each treatment. These test media (control, solvent control (spiked with 100 µl DMF/l), and test item treatment (0.20 mg test item/l; 100 µl DMF/l)) were inoculated with the same amount of algae as the test media used for the toxicity testing. At the end of the test, the treatment replicates used in the test were pooled for measurement.

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 medium was also recorded daily.


TEST CONCENTRATIONS

The study design was based on the results of range-finding pre-tests (non-GLP). The results of the test were not reported.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Results with reference substance (positive control):
The result of the latest positive control test performed in October 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.99 mg/l (study C97564), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71 - 1.7 mg/l).
Reported statistics and error estimates:
The 72-hour EC50 values could not be calculated due to the absence of a toxic effect. For the determination of the NOEC, the average growth rate and yield at the test concentration were compared to the values in the solvent control by Dunnett t test

Table 1 Biomass of Algae

 

Nominal test substance concentration

Initial measured concentration

Geometric mean measured concentration

Rep. no.

Biomass of algae*

(mg/l)

(mg/l)

(mg/l)

24 hours

48 hours

72 hours

0 (Solvent control)

---

---

1

9.1

42.3

198.7

2

8.4

45.0

192.1

3

10.5

48.6

181.7

4

9.6

45.0

177.6

5

9.0

44.9

188.0

6

9.4

45.7

172.6

Mean

9.3

45.3

185.1

SD

0.7

2.0

9.7

0 (Control)

---

---

1

9.1

46.6

173.7

2

9.2

45.1

179.7

3

8.9

45.3

179.7

Mean

9.1

45.7

177.7

SD

0.2

0.8

3.5

0.20

0.13

0.019

1

8.7

44.5

168.9

2

8.6

41.7

169.7

3

9.2

42.7

212.0

4

8.3

42.0

161.4

5

8.3

41.2

158.2

6

7.9

50.2

161.8

Mean

8.5

43.7

172.0

SD

0.4

3.4

20.1

 

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 1.43 x 103 relative fluorescence units.

Table 2 Average Growth Rates(µ)

 

Nominal
test substance concentration

Initial measured
concentration

Geometric mean measured concentration

Average growth rate µ (day-1) and inhibition of µ (Ir)

0-24 h

0-48 h

0-72 h

(mg/l)

(mg/l)

(mg/l)

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

0 (Solvent Control)

---

---

1.87

0.0

1.73

0.0

1.62

0.0

0 (Control)

---

---

1.84

1.5

1.73

-0.3

1.61

0.8

0.20

0.13

0.019

1.78*

4.9

1.71

1.1

1.59

1.6

 

*:     Mean value significantly lower than in the solvent control (according to Dunnett t-test, one-sidedsmaller,a= 0.05)

 

 

Table 3 Yield(Y)

 

Nominal
test substance concentration

Initial measured
concentration

Geometric mean measured concentration

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

0-24 h

0-48 h

0-72 h

(mg/l)

(mg/l)

(mg/l)

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

0 (Solvent Control)

---

---

7.9

0.0

43.8

0.0

183.7

0.0

0 (Control)

---

---

7.6

3.5

44.2

-0.9

176.3

4.0

0.20

0.13

19

7.1*

10.5

42.3

3.6

170.6

7.1

 

*:       Mean value significantly lower than in the solvent control (according to Dunnett t-test, one-sidedsmaller,a= 0.05)

 

 

Table 4 Section-by-Section Growth Rates

 

Nominal
test item concentration

Initial measured
concentration

Mean measured concentration

Section-by-section growth rates (day-1)
and inhibition of the growth rates (Ir)

0-24 h

24-48 h

48-72 h

(mg/l)

(mg/l)

(mg/l)

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

0 (Solvent Control)

---

---

1.87

0.0

1.58

0.0

1.41

0.0

0 (Control)

---

---

1.84

1.5

1.62

-2.4

1.36

3.5

0.20

0.13

19

1.78

4.9

1.64

-3.5

1.37

2.9

 

 

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >0.019 mg/l and NOEC of ≥0.019 mg/l have been determined for the effects of the test substance on growth rate and yield of Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata) based on geometric mean measured concentrations. The water solubility limit of the test substance has been determined in a separate study to be 0.020 mg/l.

Description of key information

Toxicity to aquatic algae: 72-h EC50: >0.019 mg/l and NOEC ≥0.019 mg/l (geometric mean measured concentrations) (highest concentration tested) (OECD Guideline 201 (Alga, Growth Inhibition Test)); growth rate and yield Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

Additional information

A 72-hour EC50 value of >0.019 mg/l and NOEC of ≥0.019 mg/l have been determined for the effects of the registered substance on growth rate and yield of Pseudokirchneriella subcapitata based on geometric mean measured concentrations.

It is likely that the test organisms were exposed to the parent substance.

The water solubility limit of the test substance has been determined in a separate study to be 0.02 mg/l.

A 72-hour EC50 of >100 mg/l and NOEC of <6.3 mg/l have been determined for the effects of trimethoxysilane (CAS: 2487-90-3) on growth rate and biomass of Pseudokirchneriella subcapitata. The test organisms will have been exposed to the hydrolysis products of the substance. Read-across of trimethoxysilane is not used to fulfil any data-gap, it is used only to demonstrate that reaction of the Si-H bond does not cause toxic effects.

Taken together these data indicate that the registered substance does not exhibit short-term toxicity to aquatic algae at its solubility limit in aquatic toxicity test media and that Si-H further reactions with electrophilic compounds are not likely to occur or do not affect short-term toxicity.