Methyl methacrylate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Methyl methacrylate
- Physical state: colourless fluid
- Analytical purity: 99.99 %
- Purity test date: 2001-09-05 (certificate of analysis AN 22216545)
- Lot/batch No.: 0071030103M
- Storage condition of test material: Storage temperature was between minimum 18°C and maximum 40°C in the dark.
- Supplier: Röhm GmbH & Co. KG, Darmstadt, Germany

Analytical monitoring:

yes

Details on sampling:

- Sampling method: From all vessels 2 samples were taken in gastight, closed HPLC vials. The samples were analysed by HPLC directly after sampling.
- Sampling frequency: Up to day 11, daily samples were taken to monitor the test concentrations during the adjustment period of the final flow rates. From day 14 to day 35, the systems were sampled two times a week.

Vehicle:

no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Source: laboratory breed. Origin of the strain of zebrafish: West Aquarium GmbH, PB 146, 37431 Bad Lauterberg, Germany. Fertilised eggs for the test were obtained from individuals which were reared in the laboratory of the Fraunhofer-Institute, Schmallenberg, Germany.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: approx. 80 fish per vessel. Holding temperature was 26 °C ± 1 °C. Light/dark cycle was 12 h/12 h. Flow through rate was adjusted to reach a 2-fold exchange of water per day. Fish were fed daily ad libitum with TetraMinR Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina).
- Method of collection of fertilised eggs: Eggs were collected with a spawning-tray (made of glass) which was placed at the bottom of the holding vessels. The tray was covered with a lattice (stainless steel) to prevent the adults from predating on the eggs, and artificial plant substrate to stimulate spawning into the tray. Mating of the fish was induced, when lighting was switched on (one fluorescent lamp per vessel, light intensity approximately 1000 lux, measured 5 cm above the water surface in the middle of the test vessel).
- Subsequent handling of eggs: The collected eggs were transferred from the spawning-tray onto a sieve, rinsed with clean water in order to remove faeces and food residuals, and then put into glass dishes. Fertilised eggs (microscopic determination of early blastula stage) were then pipetted (widened and de-burred pipette tip) into the test solutions. Time from spawning until transferring into the test solutions did not exceed two hours.


POST-HATCH FEEDING
- Start date: from day 6
- Type/source of feed: from day 6 on with breeding food (Tetra, AZ 000). From day 9 on, brine shrimp nauplii (Artemia salina). From day 16 on ground TetraMin flake food was added to the daily food.
- Amount given: ad libitum
- Frequency of feeding:daily

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Test temperature:

25.7 (±0.7) °C

pH:

6.5 - 8.2 (depending on test concentrations)

Nominal and measured concentrations:

Test concentrations (nominal): 9.38, 18.8, 37.5, 75.0,  and 150 mg/L

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Fertilised eggs were kept in plastic cages with nylon nets forming the bottom of the cages.
- Test vessel:
- Type (delete if not applicable): closed by plexiglass covers
- Material, size, headspace, fill volume: full glass aquaria of 30 x 22 x 24 cm (l x w x h) with a total volume of 15.8 litres and approx. 10.0 litres of test solution
- Aeration: no
- Type of flow-through: Dilution water was pumped by membrane pumps for each vessel. Adequate amounts of stock solution were added via a 12 channel peristaltic pump. The test substance solution was introduced beneath the water surface.
- Renewal rate of test solution: 12 volumes per day
- No. of fertilized eggs/embryos per vessel: 100 fertilised eggs
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified drinking water according to OECD-guideline The purification included filtration with activated charcoal, passage through a lime-stone column and aeration. To avoid copper contamination, plastic water pipes are used for the testing facilities.
- Intervals of water quality measurement: pH-value, oxygen concentration, and temperature were measured directly before adding the fish and afterwards daily up to day 11 and in the following period twice a week until study termination. Minimum and maximum values of the water temperature were measured continuously and recorded together with the other measurements.


OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle of 12 h/12 h


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Endpoints: mortality/survival rate, hatching rate, length and weight. After 6, 14, 21, and 35 days larvae/juvenile fish were photographed (digital camera: Olympus C 1400 L) and the survival rates (each date) as well as the lengths (at day 35, only) of the animals were determined using digital image processing (UTHSCSA ImageTool Version 2.0, Alpha 2; University of Texas Health Science Center at San Antonio, 1997).

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

9.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatching success, length and weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

18.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatching success, length and weight

Duration:

35 d

Dose descriptor:

LC10

Effect conc.:

16.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

35 d

Dose descriptor:

LC50

Effect conc.:

33.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Methyl methacrylate affected survival at concentrations equal to or  higher than 18.8 mg/L mainly at the life stage before, during and  directly after hatch (within the first 6 days). At the same  concentrations, growth was reduced significantly. Thus, after 35 days  (study termination), the NOEC (based on nominal concentrations) was  observed at 9.4 mg/L, the LOEC at 18.8 mg/L, the LC50 (based on  time-weighted averages) was calculated with 33.7 mg/L (c.l. 30.7-37.1 mg/L), the LC10 with 16.9 mg/L.

Reported statistics and error estimates:

Statistical analysis
Statistical analysis of the toxicological endpoints was conducted using the program ToxRat Professional. The calculations of LC50-values (survival rates) were based on probit analysis. Mortality in controls were included by application of Abbott's formula and effects of 0 or 100 % were substituted by 0.1 and 99.9 %, respectively. This substitution was necessary to permit the use of data of all treatment levels. Determination of No Observed Effect Concentrations (NOEC) and Lowest Observed Effect Concentrations (LOEC) was performed using Williams' test with a one-sided significance level of 0.05. The residual variance of an ANOVA was applied in the test. For survival rates, the data were expressed as p values (100 % = 1) and arcsin√(p)-transformed prior to further statistical analysis.

Results of the analytical measurements of the test concentration:

	Nominal concentrations (mg/L)
	9.4	18.8	37.5	75	150
Total mean	9.04	22.1	50.0	114	214
% of nominal	96	118	133	152	143

Hatching success and survival up to day 6, post hatch success and total success (%) up to day 6

	Nominal concentration (mg/L)
success	Control		9.4		18.8		37.5		75		150
Hatch	95	94	91	92	78	85	60	68	0	0	0	0
Post-hatch	86	83	88	83	90	79	37	37
Total	80	77	78	75	69	66	21	24	0	0	0	0

Weights and mean lengths at study termination

	Nominal concentration (mg/L)
	control		9.4		18.8		37.5
Group weights (g)	1.240	1.100	1.240	1.325	0.885	0.873	0.270	0.282
Weight per fish (mg)	15	14	16	17	13	13	12*	11*
Mean lenght (cm)	1.164	1.031	1.150	1.120	0.936*	0.993*	0.840*	0.883*

 significance p<0.05

Validity criteria fulfilled:

yes

Conclusions:

Methyl methacrylate was tested ín a fish early life stage test acc. OECD 210 under flow through conditions with Danio rerio at nominal test concentration of  9.38, 18.8, 37.5, 75.0,  and 150 mg/L. After  35 days, the NOEC (nominal conc.) was  9.4 mg/L based on hatching, weight and length.

Executive summary:

Methyl methacrylate was tested ín a fish early life stage test acc. OECD 210 under flow through conditions with Danio rerio at nominal test concentration of  9.38, 18.8, 37.5, 75.0,  and 150 mg/L. After  35 days, the NOEC (nominal conc.) was  9.4 mg/L based on hatching, weight and length, the LOEC (hatch, weight and length) was 18.8 mg/.

LC50 and LC10 based on mortality were 33.7 mg/L and 16.9 mg/L, respectively.

Description of key information

The chronic NOEC (35d) of MMA in fish is 9.4 mg/L (basis of effect: hatching, weight and length).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

9.4 mg/L

Additional information

In an Early Life Stage study with Danio rerio as test organism according to OECD guideline 210 a NOEC of 9.4 mg/L was determined. The LOEC and LC50 were measured to be 18.8 mg/L and 33.7 mg/L, respectively.