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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:
2002-01-24 to 2002-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 50 mg/l stock solution was prepared by placing 0.049 ml (density of 1.028 g/ml) of aminosilane in a 1000 ml volumetric flask and diluting to volume with sterile AAP medium containing 0.10 ml/l of dimethyl formamide (DMF, CAS No. 68-12-2).  The resulting stock solution was observed to be clear and colourless with no visible undissolved test substance.  Nominal test concentrations were prepared from dilutions of the 50 mg/l stock solution. A solvent control was prepared with AAP medium containing 0.1 ml/l of DMF. A blank control comprising only of AAP medium was also prepared.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Strain: 1648

- Source (laboratory, culture collection): The alga was obtained from Carolina Biological Supply Co., Burlington, North Carolina, and was maintained in stock culture at Springborn Smithers. 

- Culture conditions: The stock cultures were maintained within the following conditions:  a shaking rate of 100 ± 10 rpm, a temperature of 24 ± 1 °C and continuous illumination at the surface of the medium with an intensity of approximately 300 to 500 footcandles (3200 to 5400 lux).  Lighting was supplied by Duro-Test® Vita-Lite® fluorescent bulbs.  Culture flasks were agitated continuously on an orbital shaker.

- Growth/test medium:  The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water.  

- Culture vessesl: Stock cultures were grown in 250 ml glass flasks, each containing 100 ml of culture medium. The flasks were closed with stainless steel caps that permitted gas exchange.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
No data but conductivity measured at start and end of test ranged between 75 and 90 μmhos/cm
Test temperature:
23 to 24 °C
pH:
7.2-8.7 at start of test

7.8-8.2 at end of test
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal test concentrations: 0 (Control), 0 (Solvent control), 1.6, 3.1, 6.3, 13, 25 and 50 mg/l
Details on test conditions:
TEST SYSTEM

- Growth/test medium:  The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water.  

- Exposure vessel type: The test was conducted in sterile 250-ml Erlenmeyer flasks containing  100-ml of test solution.  All test vessels were fitted with stainless steel caps which permit gas exchange.  

- Culture medium:  The culture mediumused was Algal assay Procedure (AAP) medium prepared with sterile deionised water. The TOC concentration of a sample collected in January 2002 was 0.47 mg/l.

- Light levels and quality during exposure:  320 - 420 footcandles (3400 - 4500 lux).  The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 50 to 69 µE/m2/s.

- Test Design:  Approximately 30 minutes after the test solutions were added to the test flasks (100 ml per flask), a 0.323-ml inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 310 x 10E4 cells/ml, was aseptically introduced into each flask.  This inoculum provided the required initial (0 hour) cell density of approximately 1.0 x 104 cells/ml.  Three replicate test vessels were established for each treatment level, the dilution water control and the solvent control.

- Determination of cell concentrations: At the start of the test and at each subsequent 24 observation period, cell counts were made on each three replicates of each treatment using a hemacytometer (Neubauer improved).

- Range finding test: The test concentrations were set on the basis of the results of a range finding test conducted at concentrations of 0.00095, 0.0095, 0.095 and 0.95 mg/l and a second test conducted at concentrations of 60, 130, 240, 480 and 960 mg/l. No effects were observed in the first test and effects at all treatment levels were observed in the second test.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Remarks on result:
other: 2.3-34 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
biomass
Remarks on result:
other: 1.8-17
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
biomass
Remarks on result:
other: determined empiracally as the highest concentration with <10% inhibition
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
other: cell density
Remarks on result:
other: 2.2-61 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
6.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
other: cell density
Details on results:
- Exponential growth in the control (for algal test): yes

- Control cell density at end of test: 1580000 cells/ml (Control), 1360000 cells/ml (Solvent control), 1470000 cells/ml (Pooled control)
Reported statistics and error estimates:
The EC50 values were determined by linear regression using the method of inverse regression.

The NOECs were determined by either Williams' test (if the data sets passed the test homogeneity and normality) or the Kruskal-Wallis'test if they did not. If the statistical analyses did not yield a reasonable NOEC it was selected empiracally as the highest treatment level that resulted in <10% inhibition.

All statistical determinations were made at 95% level of certainty.

Table 1. Test results

 Nominal concentration (mg/l)

 Mean cell concentration after 24 hours (cells/ml)

  Mean cell concentration after 48 hours (cells/ml) 

  Mean cell concentration after 72 hours (cells/ml) 

  Mean cell concentration after 96 hours (cells/ml) 

Percentage reduction in cell concentration at end of the test* 

Percentage reduction in biomass at end of the test* 

Percentage reduction in 0 -72 -hour growth rate*

0 (Control)

24200

75000

435000

1580000

-

-

-

0 (Solvent control)

21700

75000

408000

1360000

-

-

-

0 (Pooled control)

20000

80000

420000

1470000

-

-

-

1.6

17500

75800

390000

1420000

3

7.0

2.0

3.1

10800

52500

413000

1590000

-8

14

1.0

6.3

7500

35800

256000

1220000

17

48

15**

13

800

10000

87000

860000

41**

89

44**

25

800

800

3000

10000

99**

108**

128**

50

800

5000

3000

0

100**

106

128**

*A negative sign (-) indicates that biomass or growth rate was higher than in the Control.

** Significantly different compared with pooled control


Validity criteria fulfilled:
yes
Conclusions:
A 96-hour EC50 value of 8.8 mg/l and NOEC of 3.1 mg/l have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata. The results are expressed relative to nominal concentrations of the test substance. However the substance is subject to rapid hydrolysis and under the test conditions it is therefore likely that exposure will have been to its hydrolysis products (methanol and trisilanols).
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:
Please refer to the attached justification for grouping of substances provided in IUCLID Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Remarks on result:
other: Source: Springborn Smithers 2002
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Remarks on result:
other: 2.3-34 mg/L
Remarks:
Source: Springborn Smithers 2002
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
352 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Remarks on result:
other: Source: Hüls 1994
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Remarks on result:
other: Source: Hüls 1994
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994-11-29 to 1994-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Cited as Directive 92/69/EEC, C.3
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The Dissolved Organic Carbon (DOC) concentration of the stock solution used to prepare the test media and the test media was determined at athe start and end of the test
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: The test substance was added to the dilution medium at 1 g/l and stirred for 18 hours. After that the solution was filtered and the DOC determined.  The stock solution was used to prepare the other treatments by dilution.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM

- Strain: 86.81.SAG

- Source (laboratory, culture collection): Institute for Water, Ground and Air Hygiene, Berlin (Germany)

- Age of inoculum (at test initiation): 3 days

- Method of cultivation: A pre-culture is produced from an original culture by super-inoculation three days before the test begins. From this culture the test cultures are inoculated at a density of about 20000 cells/ml.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
24 +/-1ºC
pH:
6.9-8.4 at start of test

8.6-9.5 at end of test
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations (based on measured DOC concentration in stock solution converted to the equivalent test substance concentration): 0 (Control), 4.4, 9.1, 20, 44, 91, 200 and 436 mg/l.

The measured concentrations at the start and end of the test were with 20% of the nominal values and therefore the test results are interpreted with reference to nominal concentrations.
Details on test conditions:
TEST SYSTEM

- Test vessel:

- Type: open Erlenmeyer flasks

- Material: glass

- Aeration: none

- Initial cells density: 20000 cells/ml

- Control end cells density: 660000 cells/ml

- No. of vessels per concentration (replicates): 5

- No. of vessels per control (replicates): 8

GROWTH MEDIUM

- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS

- Culture medium different from test medium: no

- Intervals of water quality measurement: start and end of test

OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: no

- Photoperiod: continuous

- Light intensity and quality: 8000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Algal cell counts were made photometrically at 0, 24, 48 and 72 hrs.

- Determination of cell concentrations: spectrophotometer (absorption at 685 nm)

TEST CONCENTRATIONS

- Spacing factor for test concentrations: 2.1-2.2
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
other: biomass and growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
352 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
126 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
EC50 values were determined by Probit Analysis

Table 1. Test results

 Nominal concentration (mg/l)  Nominal cell concentration at start of test (cells/ml)  Mean cell concentration after 24 hours (cells/ml)   Mean cell concentration after 48 hours (cells/ml)    Mean cell concentration after 72 hours (cells/ml)  Percentage reduction in biomass at end of the test*  Percentage reduction in 0 -72 -hour growth rate*
0 (Control) 20000 80000 230000 660000  -  -
4.4 20000 60000 170000 740000 6.8 -3.3
9.1 20000 70000 210000 850000 -11.0 -7.2
20 20000 60000 200000 900000 -3.4 -5.5
44 20000 70000 170000 540000 22.0 5.7 
91 20000 70000 130000 310000 48.3 21.6 
200 20000 60000 100000 180000 66.1 37.2
436 20000 60000 60000  110000 78.8 51.3

*A negative sign (-) indicates that biomass or growth rate was higher than in the Control.

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of 352 mg/l and NOEC of 20 mg/l have been determined for the effects of the test substance on growth rate of Scenedesmus subspicatus (new name: Desmodesmus subspicatus). The results are expressed relative to nominal concentrations of the test substance. However the substance is subject to rapid hydrolysis and under the test conditions it is therefore likely that exposure will have been to its hydrolysis products (methanol and trisilanols).

Description of key information

key study (1): 72-hr EC50 of 8.8 mg/l and NOEC of 3.1 mg/l (Selenastrum capricornutum new name Pseudokirchnerella subcapitata; OECD 201), RL1

Key value for chemical safety assessment

Additional information

Measured data was not available for N-[3-(dimethoxymethylsilyl)-2-methylpropyl]ethylenediamine (CAS 023410-40-4), therefore read across from the comparable substance, N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3) was deemed acceptable for assessment.

A 72-hour EC50 value of 8.8 mg/l and NOEC of 3.1 mg/l have been determined for the effects of the test substance on growth rate of Pseudokirchneriella subcapitata. The results are expressed relative to nominal concentrations of the test substance. However the substance is subject to rapid hydrolysis and under the test conditions it is therefore likely that exposure will have been to its hydrolysis products (methanol and N-(3-(trihydroxysilyl)propyl)ethylenediamine). This result has been selected as the lowest EC50 value for effects on growth rate from four reliable studies. The other reliable EC50 value was 352 mg/l and the corresponding NOEC is 20 mg/l.

The results are expressed relative to nominal concentrations of the test substance. However the substance is subject to rapid hydrolysis and under the test conditions it is therefore likely that exposure will have been to its hydrolysis products (methanol and trisilanols).