Trimethyl orthoacetate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: ATCC-22662
- Source (laboratory, culture collection): National Institute Environmental Research, Korea
- Age of inoculum (at test initiation): 7 days
- Method of cultivation: The flasks then were placed on a shaker incubator at 170 rpm and 25°C, with 24 h light supplied via worm white fluorescent tubes, with an average illumination of 30 ± 5µE/m2s.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Stock alga was cultivated in 250 mL Erlenmeyer flasks, containing 200 mL sterilized nitrate-enriched BBM medium prepared in triple distilled water, to avoid nitrogen limitation during the high-density culture. The culture flask was agitated on a shaker at 170 rpm, and bubbled with air (1 vvm), without sparger. Light was continuously supplied, with an average of 30 ±5 µE/m2s, using warm-white fluorescent tubes located on top of the shaker. All the flasks were maintained in the shaker incubator at 25 ± 5°C for 7 days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

25°C

Details on test conditions:

TEST SYSTEM

Experiments were performed in 250 mL Erlenmeyer flasks containing 55 mL of sterilized culture medium, inoculated with 5 mL samples of 7 day cultured algae. Solutions of organic solvents were subsequently added to the test flasks. The organic solvents tested were completely miscible with water, forming a homogeneous phase. Concentrations were adjusted in the range between 0.1 M and 1.26 µM to obtain complete concentration-response curves. At each determined exposure date, the optical density of the algal biomass was estimated at 438 nm using a spectrophotometer. The dry cell weight, corresponding to the optical density, was determined from the linear relationship; dry cell weight (g/L) = 0.329 x optical density. All experiments were performed in duplicate, but the control experiments were conducted in triplicate.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 22 000 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

growth rate

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Growth inhibition test

GLP compliance:

not specified

Analytical monitoring:

not specified

Test organisms (species):

other: Anabaena sp., A. cylindrica, A. variabilis, A. inaegualis, and Nostoc sp.

Details on test organisms:

TEST ORGANISM
- Common name: blue-green algae
- Strain and Source: Anabaena sp. ATCC 27899, A. cylindrica ATCC 29414, A. variabilis ATCC 29413, and Nostoc sp. ATCC 29105 were obtained from the American Type Culture Collection, Rockville, Maryland, U.S.A., A. inaequalis was obtained from the Department of Botany and Genetics, University of Guelph, Guelph, Ontario, Canada.
- Method of cultivation: Stock cultures were grown in 250 mL Erlenmeyer flasks sealed with cotton bungs and containing 150 mL of an inorganic, nitrogen-free medium (Stratton et al. 1980). Flasks were incubated at 25 ± 1°C and a light intensity of 7 klux on a 12 hour light-dark cycle.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Nominal and measured concentrations:

A minimum of 10 concentrations were tested for each species, ranging from 0.6% to 10% methanol by volume (4.74-79.00 g/L).

Details on test conditions:

TEST SYSTEM
- No. of vessels per concentration (replicates): 5 - 10
- experiment repeated 3 - 5 times

Reference substance (positive control):

no

Key result

Dose descriptor:

EC50

Effect conc.:

20 300 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

other: growth inhibition

Remarks on result:

other: Anabaena cylindrica

Key result

Dose descriptor:

EC50

Effect conc.:

21 170 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

other: growth inhibition

Remarks on result:

other: Anabaena inaequalis

Key result

Dose descriptor:

EC50

Effect conc.:

24 730 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

other: growth inhibition

Remarks on result:

other: Anabaena sp.

Key result

Dose descriptor:

EC50

Effect conc.:

43 290 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

other: growth inhibition

Remarks on result:

other: Nostoc sp.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22 April - 29 April, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant. "Based on" a standard guideline. Limitations on reporting. Available as an unpublished report. Acceptable with restrictions.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Principles of method if other than guideline:

Test performed according to lab protocol which is "based on" ISO guideline.
72 h growth inhibition test on Skeletonema costatum, measuring growth rate in order to identify inhibition.

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance dissolved in test medium to form stock solution of the highest test dosage, mixed with magnetic stirrer for 30 minutes. Test series prepared from this by dilution.
- Controls: Untreated test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA BAC 1

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

Within 20.0 ± 2.4 °C

pH:

Test start: 8.29
Test end: 8.38
Measured in 1000 mg test item/L solution.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Control, 10, 50, 250 and 1000 mg test item/L.
All values nominal.

Details on test conditions:

TEST SYSTEM
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Algal medium 1/2 "f" from Guillard and Ryther (1962) was used.


OTHER TEST CONDITIONS
- Light intensity and quality: 6000-10000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence (Turner 10-100 R fluorometer)
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (1.5 mg/L)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 300.82 mg/L

Nominal / measured:

nominal

Conc. based on:

other: acetate ion

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

300.82 mg/L

Nominal / measured:

nominal

Conc. based on:

other: acetate ion

Basis for effect:

growth rate

Details on results:

No inhibition of the growth rate observed in any of the test concentrations or the control.

Results with reference substance (positive control):

33 % inhibition.

Reported statistics and error estimates:

Data processed by EXCEL.

Table 1. Results

Dosage (mg test item/L)	Growth Rate		% Inhibition
	Mean	Confidence limit	Mean	Confidence limit
Control	1.56	0.02	-	-
10	1.56	0.01	-1.1	0.9
50	1.57	0.02	-1.7	1.3
250	1.57	0.03	-1.4	1.7
1000	1.54	0.04	0.2	2.4

Validity criteria fulfilled:

yes

Remarks:

Control cell density increase by > 16 at 72 h. Control pH varies by < 1.0 units over course of test. Temperature within range 20.0 ± 2.4°C. Light intensity within 6000-10000. Inhibition of growth rate with 1.5 mg 3,5-dichlorophenol/L between 20 and 80%.

Conclusions:

The 72hr EC50 for Skeletonema costatum was calculated to be >300.82 mg/l based on the effect of the acetate ion.

Executive summary:

In a GLP compliant, guideline 72 hr acute toxicity test of the test substance to the marine algal species Skeletonoma costatum, no significant effect was seen up to the highest test concentration of 1000 mg/L. Therefore the EC50 was determined to be >1000 mg/L for the test substance. The 72hr EC50 for Skeletonema costatum was calculated to be >300.82 mg/l based on the effect of the acetate ion.

The reported EC50 was recalculated in order to report the effect concentrations based on the concentration of the acetate ion. The EC50 was adjusted for the molecular weight of the acetate compared to the parent molecule and also the fact that the test substance is only 50% potassium acetate.

Although it is prefereable to have data from tests on freshwater algal species, this study is considered suitable for use as a key study as the results are the same as for fish and Daphnia.

Description of key information

Trimethyl orthoacetate is rapidly hydrolysed to methanol (CAS no. 67 -56 -1) and methyl acetate, which further hydrolyses to acetic acid (CAS no. 64 -19 -7) as final reaction product in the presence of water (< 2.4 h at pH 4, 7 and 9 at 50°C). Under neutral (pH 7) and acidic (pH 4) conditions, the half-life was < 1 h. The measured degradation of TMOA at pH 7 abd 50°C correspond to a half-life time well below one day under normal outdoor conditions. Accordingly, reliable data of the hydrolysis products methanol and acetic acid are used to address the endpoint, which is entirely appropriate to draw conclusions on the toxicity of Trimethyl orthoacetate to aquatic algae and cyanobacteria.

1. Methanol

- Toxicity to aquatic algae and cyanobacteria: EC50 (96 h) = 22000 mg/L based on growth rate for the freshwater green-algae Pseudokirchneriella subcapitata (OECD TG 201 and EPA OPPTS 850.5400)

- Toxicity to aquatic algae and cyanobacteria: EC50 (14 d) = 20 300 mg/L based on growth inhibition for the freshwater blue-green algae Anabaena cylindrica (no guideline followed, no GLP)

2. Acetic Acid

- Toxicity to aquatic algae and cyanobacteria: EC50 (72 h) > 300.82 mg/L, NOEC (72 h) = 300.82 mg/L based on the effect of the acetate ion for the parameter growth rate for the saltwater green-algae Skeletonema costatum (ISO 10253, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

22 000 mg/L

EC50 for marine water algae:

300.82 mg/L

EC10 or NOEC for marine water algae:

300.82 mg/L

Additional information

Concerning study data for acetic acid:

In a GLP compliant, guideline 72 h acute toxicity test of the test substance to the marine algal species Skeletonoma costatum, no significant effect was seen up to the highest test concentration of 1000 mg/L (Beslagic, 2005). Therefore the EC50 was determined to be >1000 mg/L for the test substance. The 72 h EC50 for Skeletonema costatum was calculated to be >300.82 mg/l based on the effect of the acetate ion.

The reported EC50 was recalculated in order to report the effect concentrations based on the concentration of the acetate ion. The EC50 was adjusted for the molecular weight of the acetate compared to the parent molecule and also the fact that the test substance is only 50% potassium acetate.

Although it is preferable to have data from tests on freshwater algal species, this study is considered suitable for use as a key study, as the results are within the same range as for freshwater studies in fish and Daphnia.