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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with national standard methods with acceptable restrictions (e.g. no analytical verification of test concentrations, non-GLP)
Qualifier:
according to guideline
Guideline:
other: German Industrial Standard DIN 38412, Part 9
GLP compliance:
no
Analytical monitoring:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Pre-culture:
- species: Scenedesmus subspicatus (renamed to Desmodesmus subspicatus), SAG 86.81
- medium: OECD-medium (OECD 201)
- temperature: 20 °C
- test flasks: 250 mL-Erlenmeyer flasks plugged with gas permeable silicon-sponge caps
- test volume: 100 mL
- flasks were incubated in an incubation chamber for 72 h
- flasks were shaken once a day to hold cells in suspension
- illumination: permanent artificial light
- light intensity: approx. 120 µE/(m²*s)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Details on test conditions:
Test-culture:
- No. of algae in test flasks at test start: 10000 cells/ml (exponential growth phase)
- test vessels: 20-mL tubes plugged with gas permeable silicon-sponge caps
- test volume: 10 mL

- stock solution of test substance:
- conductivity: 802 µS/cm
- pH 6.64
- stock solution was diluted in test medium to reach the final test concentrations

- test concentrations: 1.954, 3.906, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 mg/L; control
- replicates: per concentration and control: 4, blank per concentration (w/o cells): 2

- tubes were incubated in an incubation chamber for 72 h at 23 °C
- tubes were shaken once a day to hold cells in suspension
- illumination: permanent artificial light
- light intensity: approx. 120 µE/(m2*s)

- samples were taken at regular intervals: 0, 24, 48, 72 h
- measurements:
- fluorescence (in vivo chlorophyll-a fluorescence at 685 nm as a size for the biomass (pulsed excitation with light flashes having a wavelength of 435 nm))
- pH
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
31.25 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
62.5 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL: 29.5-66.3 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
98.9 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL: 72.9-123.4 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
392.2 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL: 320.9-510.6 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.812 mg/L
Nominal / measured:
nominal
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
15.625 mg/L
Nominal / measured:
nominal
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
26.6 mg/L
Nominal / measured:
nominal
Basis for effect:
biomass
Remarks on result:
other: 95 % CL: 12.1-41.2 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
46.3 mg/L
Nominal / measured:
nominal
Basis for effect:
biomass
Remarks on result:
other: 95 % CL: 26.5-64.7 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
132.9 mg/L
Nominal / measured:
nominal
Basis for effect:
biomass
Remarks on result:
other: 95 % CL: 101.4-176.5 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
31.25 mg/L
Nominal / measured:
nominal
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
62.5 mg/L
Nominal / measured:
nominal
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25.7 mg/L
Nominal / measured:
nominal
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL: 5.2-45.4 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
41.2 mg/L
Nominal / measured:
nominal
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL: 13.4-64.8 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
101.5 mg/L
Nominal / measured:
nominal
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL: 64.5-160.4 mg/L

Table 1: pH-values at beginning and 72 hours respectively

Test item concentration (mg/L; nominal)

pH after x hours

(w/o algae)

pH after xx (h)

(with algae)

0 hours

72 hours

72 hours

Control

8,26

8.3

9.34

500

8.01

7.83

8.08

250

8.12

7.89

8.33

125

8.21

7.88

8.63

62.5

8.25

7.90

8.94

31.25

8.28

7.89

9.37

15.625

8.34

7.93

9.51

7.812

8.48

7.94

9.51

3.906

8.48

8.01

9.40

1.953

8.44

8.10

9.32

Table 2: Results of inhibiton

Treatment [mg/L]

Growth rate inhibition [%]

Biomass decrease [%]

Yield Decrease [%]

Control

0.0

0.0

0.0

1.954

-0.6

-0.1

-1.9

3.906

-1.7

-1.3

-5.3

7.812

-1.8

-2.3

-3.6

15.625

0.5

3.3

1.9

31.250

0.5

2.8

2.1

62.500

13.8

29.8

40.0

125.00

27.1

54.5

62.4

250.00

42.5

72.8

77.8

500.00

52.1

74.7

84.4

Validity criteria fulfilled:
yes
Conclusions:
As final results, an EC 10 (72 h) of 26.6 mg/L and an EC 50 (72 h) of 132.9 mg/L were determined based on biomass.
Furthermore, an EC 10 (72 h) of 48 mg/L and an EC 50 (72 h) of 392.2 mg/L were calculated based on growth rate. These values refer to the nominal test concentrations.
Executive summary:

The toxicity of the test substance trimethylamine hydrochloride (CAS 593 -81 -7) towards Algae has been investigated according to DIN 38412, part 9. GLP compliance is not given. As test organism, Scenedesmus subspicatus (new name: Desmodesmus subspicatus) has been used (Strain: 86.81). The total exposure duration amounts to 72 hours. Nominal test concentrations of 1.954, 3.906, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 mg/L have been used (no analytical verification). An initial cell density of 10 000 cells/mL was applied. Four test vessels (20 mL tubes plugged with gas permeable silicon-sponge caps) per concentration as well as for the control have been used, respectively. Two basis for effects were chosen: the growth rate and the biomass. Cell concentrations were determined using a fluorimeter after 0, 24, 48 and 72 h.

As final results, an EC 10 (72 h) of 26.6 mg/L and an EC 50 (72 h) of 132.9 mg/L were determined based on biomass.

Furthermore, an EC 10 (72 h) of 48 mg/L and an EC 50 (72 h) of 392.2 mg/L were calculated based on growth rate. These values refer to the nominal test concentrations.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, no GLP, no analytical monitoring
Qualifier:
according to guideline
Guideline:
other: DIN 38412, part 9
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: 86.81

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
20 °C
pH:
uninoculated: 8.1 - 8.5
inoculated: 8.5 - 9.9
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Nominal test concentrations: 7.81, 15.83, 31.25, 82.5, 125, 250, 500 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (250 mL) plugged with gas permeable silicon-sponge caps
- Type (delete if not applicable): open
- Material: glass
- Fill volume: 100 mL
- Initial cells density: 10 000 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels uninoculated control (replicates): 6
- No. of vessels control (replicates): 1
- medium: OECD-medium
- flasks were incubated in an incubation chamber for 72 h
- flasks were shaken once a day to hold cells in suspension
- illumination: permanent artificial light
- light intensity: approx. 120 µE/(m2*s)

Preparation of stock solution:
- 1 g test substance/litre (sterile bidest. water; test substance is volatile; fluid appeared clear; conductivity: 13 µS/cm, pH 10.2); stock solution was diluted in test medium to reach the final test concentrations.
- tubes were shaken once a day to hold cells in suspension
- illumination: permanent artificial light
- light intensity: approx. 120 µE/(m2s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter (in vivo chlorophyll-a fluorescence at 685 nm as a size for the biomass (pulsed excitation with light flashesurements) after 0, 24, 48, 72 and 96 h
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
150 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
recalculated
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
86 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
recalculated
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
90.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
recalculated
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
42.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
recalculated

During the 96 h exposure to trimethylamine fluorescence values (given as % of control) were as follows (average of 6 replicates):

Table 1: Fluorescence values (given as % of control

Nominal concentration (mg TMA/L)  24 h  48 h  72 h   96 h
7.81 109.92 111.63    130.69    108.36
15.83

 102.52

89.67 

  108.69

98.84

31.25

 93.01

96.3

  112.76

  87.79

82.5

  72.93

 97.84

  74.17

   54 .96

125

42.28

 37.17

    32.54

   28.37

 250

11.63

 1.82

  0.55

  0.7

 500

0

-0.13 

 0

   -0.02

Table 2: pH values

 Nominal test

concentrations (TMA)

test start;

not inoculated

 96 h; 

not inoculated

   96 h;

inoculated

 control  8.0  8.2  9.8
 7.81  9.7  8.5  8.5
 15.63  9.4  8.4  8.5
 31.25  9.1  8.3  9.0
 62.5  8.8  8.2  9.5
 125  8.5  8.2  9.9
 250  8.3  8.1  9.9
 500  8.1  8.2  9.9

Table 3. inhibition results

Treatment [mg TMA/L]

Growth rate inhibition [%]

Biomass decrease [%]

Yield Decrease [%]

Control

0.0

0.0

0

7.81

-5.9

-23.3

-31.1

15.63

-1.4

-1.5

-8.8

31.25

-4.0

-5.8

-13.0

62.50

6.5

29.2

26.1

125

26.0

67.8

68.4

250

122.8

102.3

100.9

500

235.8

103.1

101.1

Validity criteria fulfilled:
yes
Conclusions:
As final results for TMA, an EC 10 (72 h) of 42.6 mg/L and an EC 50 (72 h) of 90.6 mg/L were determined based on biomass. Furthermore, an EC 10 (72 h) of 86 mg/L and an EC 50 (72 h) of 150 mg/L were calculated for TMA based on growth rate. These values refer to the nominal test concentrations.
Executive summary:

The toxicity of the test substance trimethylamine (CAS 75-50-3) towards Algae has been investigated according to DIN 38412, part 9. GLP compliance is not given. As test organism, Scenedesmus subspicatus (new name: Desmodesmus subspicatus) has been used (Strain: 86.81). The total exposure duration amounts to 96 hours. Nominal test concentrations of 7.81, 15.83, 31.25, 82.5, 125, 250 and 500 mg/L have been used (no analytical verification). An initial cell density of 10 000 cells/mL was applied. Six test vessels (Erlenmeyer flasks, 250 mL) per concentration as well as for the uninoculated control have been used, respectively. One test vessel has been used for the control. Two basis for effects were chosen: the growth rate and the biomass. Cell concentrations were determined using a fluorimeter after 0, 24, 48, 72 and 96 h.

As final results for TMA, an EC 10 (72 h) of 42.6 mg/L and an EC 50 (72 h) of 90.6 mg/L were determined based on biomass. Furthermore, an EC 10 (72 h) of 86 mg/L and an EC 50 (72 h) of 150 mg/L were calculated for TMA based on growth rate. These values refer to the nominal test concentrations.

Description of key information

Trimethylamine hydrochloride_DIN 38412, part 9_Desmodesmus subspicatus_EC10 (72 h) = 48 mg/L and EC50 (72 h) = 392.2 mg/L (both based on growth rate)

Trimethylamine (read across)_DIN 38412, part 9_Desmodesmus subspicatus_EC10 (72 h) = 86 mg/L and EC50 (72 h) = 150 mg/L (both based on growth rate)

Key value for chemical safety assessment

EC50 for freshwater algae:
392.2 mg/L
EC10 or NOEC for freshwater algae:
48 mg/L

Additional information

The toxicity of the test substance Trimethylamine hydrochloride (CAS 593 -81 -7) towards Algae has been investigated according to DIN 38412, part 9. GLP compliance is not given. As test organism, Scenedesmus subspicatus (new name: Desmodesmus subspicatus) has been used (Strain: 86.81). The total exposure duration amounts to 72 hours. Nominal test concentrations of 1.954, 3.906, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 mg/L have been used (no analytical verification). An initial cell density of 10 000 cells/mL was applied. Four test vessels (20 mL tubes plugged with gas permeable silicon-sponge caps) per concentration as well as for the control have been used, respectively. Two basis for effects were chosen: the growth rate and the biomass. Cell concentrations were determined using a fluorimeter after 0, 24, 48 and 72 h.

As final results, an EC 10 (72 h) of 26.6 mg/L and an EC 50 (72 h) of 132.9 mg/L were determined based on biomass. Furthermore, an EC 10 (72 h) of 48 mg/L and an EC 50 (72 h) of 392.2 mg/L were calculated based on growth rate. These values refer to the nominal test concentrations.

In addition, the toxicity of the read-across substance trimethylamine (CAS 75-50-3) towards Algae has been investigated according to DIN 38412, part 9 (Supporting study 1, 1988; 2002; 2008). GLP compliance for this study is not given. As test organism, Scenedesmus subspicatus (new name: Desmodesmus subspicatus) has been used (Strain: 86.81). The total exposure duration amounts to 96 hours. Nominal test concentrations of 7.81, 15.83, 31.25, 82.5, 125, 250 and 500 mg/L have been used (no analytical verification). An initial cell density of 10 000 cells/mL was applied. Six test vessels (Erlenmeyer flasks, 250 mL) per concentration as well as for the uninoculated control have been used, respectively. One test vessel has been used for the control. Two basis for effects were chosen: the growth rate and the biomass. Cell concentrations were determined using a fluorimeter after 0, 24, 48, 72 and 96 h.

As final results, an EC 10 (72 h) of 42.6 mg/L and an EC 50 (72 h) of 90.6 mg/L were determined based on biomass. Furthermore, an EC 10 (72 h) of 86 mg/L and an EC 50 (72 h) of 150 mg/L were calculated based on growth rate. These values refer to the nominal test concentrations.

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium cation (common "breakdown product"). Both respective counterions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions. Besides the influence on the pH value of an aqueous solution (OH-), they do not bear a relevant intrinsic property, allowing one in general to focus on the Trimethylammonium cation. Toxicity of the respective counterion is not applicable (Cl-) in the aqueous environment, a slightly aggravating effect is expected for the hydroxyl anion (OH-). So primarily the trimethylammonium cation has to be evaluated. It is believed to be act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms, like algae and cyanobacteria. Therefore both substances are expected to follow the same pattern of toxicity to algae and cyanobacteria. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).