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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP In parts of the study which were not critical for the evaluation the variation of test substance concentrations exceeded ± 20 %.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Methacrylic acid is the hydrolysis product of Methacrylic anhydride. One molecule of methacrylic anhydride willl hydrolyse into two molelules methacrylic acid.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance: Methacrylic acid, purity >= 98 %
Target substance: Methacrylic anhydride: purity: >=94 %, other components: Methacrylic acid and adducts of methacrylic aanhydride and adducts of methacrylic acid and methacrylic anhydride.

3. ANALOGUE APPROACH JUSTIFICATION
Like other acid anhydrides, methacrylic anhydride was found to be hydrolytically unstable in water at pH 4, 7 and 9. The half lives (DT50) were found to be 56.4 min at pH 4 (25 °C), 25.5 min at pH 7 (25 °C) respectively 10.3 min at 37 °C and 2.29 min at pH 9. The initial hydrolysis product is methacrylic acid (Noack 2012).

4. Data Matrix
Target: Methacrylic anhydride CAS 760-93-0
Source: Methacrylic acid CAS 79-41-4
Reason / purpose:
read-across source
Specific details on test material used for the study:
Methacrylic acid (CAS: 79-41-4)
Purity: 99.79 %
Water: 605 ppm
Inhibitor: 200 ppm MEHQ
2 ppm HQ
Lot Number: 32118825
Supplier: Röhm GmbH & Co. KG
Colorless liquid with pungent odor
Analytical monitoring:
yes
Details on sampling:
12 measurements during the course of the test in intervals of 1-5  days
Vehicle:
no
Details on test solutions:
Appropriate amounts of MMA were dissolved directly in dilution water in  order to prepare the stock solution for the flow-through test.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Dano rerio (Hamilton-Buchanan), origin: West Aquarium GmbH, Bad  Lauterberg, Germany, continuously bred at Fraunhofer Institut für  Molekularbiologie und Angewandte Ökologie.  Holding: Parental fish were held in aquaria with a total volume of 150 l.  Holding water was of the same quality as used in the test (see: dilution  water). At the time of reproduction, parental fish were about 18 months  old (maximum age for parental fish is 2 years). Stock density was 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 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).  The broodstock was visually checked every day for mortality, illness,  parasites or abnormal behaviour. No prophylactic treatment of fish took  place. Only healthy fish without diseases and abnormalities were used as  parental fish for the production of fertilised eggs. Fertilisation rate  was checked in advance and had to be at least 50 % for accepting the  batch as parental fish for a study.
Breeding: Eggs were collected with a glass spawning-tray 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 (modified method according to Nagel, 1986) to stimulate spawning into the tray. 
Handling of embryos and larvae: The collected eggs were transferred from the spawning-tray onto a sieve,  rinsed with clean water in order to remove faeces and rests of food, and  then put into glass dishes. Fertilised eggs (microscopic determination of  early blastula stage) were then pipetted (widened and de-burred pipette  tip) into holding cages the test solutions. Time from spawning until  transferring into holding cages (see test conditions) did not exceed one  hour.  On day 28 the holding cages were removed and subsequently the larvae were  swimming free in the aquaria.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Hardness:
0.6-0.7 mmol/l
Test temperature:
25 °C ±2 °C
pH:
7.6-8.1
Dissolved oxygen:
10.1-11.7
Nominal and measured concentrations:
Nominal test concentrations: 0, 2.0, 4.5, 10, 23 and 51 mg/L
The mean measured concentrations were within a range of ± 10 % of the  nominal values for the higher four concentrations and within a range of ±  20 % of the nominal values for the lowest concentration. Hence, effect  concentration calculations were based on nominal values. See Results for additional details.
Details on test conditions:
Fertilized eggs of Zebrafish (Danio rerio) were exposed to five  concentrations of the test substance under flow-through conditions for a  period of 35 d. Hatching rate, mortality of embryos, yolk sac larvae,  free feeding larvae and juvenile fish as well as lengths and group  weights at the end of the test were determined. The test was performed in  accordance with the OECD guideline 210.

Test vessels: These were glass aquaria (29 x 21 x 23 cm) containing  approx. 11.0 litres of test solution. Fertilised eggs were kept in  stainless steel holding cages with nylon nets forming the bottom of the  cages.
Flow-through system: Five concentrations of MAA and the untreated control  were tested under flow-through conditions at 25°C ± 1°C and a light/dark  cycle of 12 h/12 h. For every test vessel a water flow rate of 3.0  litre/h was adjusted, resulting in a daily turnover of 6.5 volumes. Test  vessels were not additionally aerated. Dilution water was pumped by  dosage pumps into mixing chambers. Adequate amounts of stock solution  were added into the mixing chambers via dosage pumps. The nominal initial  test concentrations were 2, 4.5, 10, 23 and 51 mg/l MAA. Controls were  kept in dilution water. Control and all test concentrations were run in 2 replicates, each. Due  to poor statistical power of the first test a second test segment was run  only at three test concentrations (4.5, 10 and 23 mg/l). The test results  of both segments are reported together. 
Test conditions, measurements: pH-value (control: pH 8.03 - 8.55; 51  mg/l: pH 6.08 - 6.89), oxygen concentration (80 - 100 % at test  initiation, in the test groups of 10 mg/l and higher a lower oxygen  saturation was observed with values >= 60 %), and temperature (mean temp.  was 25.0 and 25.7 °C during  the two separate test segments; ± 1°C);  Measurements were performed directly before adding the fish and  afterwards twice a week. Minimum and maximum values of the water  temperature were checked twice a week.

A range-finding test was not performed.
Reference substance (positive control):
no
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Remarks on result:
other: mean measured conc. +/- 10 % for the four higher conc. and +/- 20 % of nominal value for the lower conc.
Key result
Duration:
35 d
Dose descriptor:
LC10
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Duration:
35 d
Dose descriptor:
LC50
Effect conc.:
42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Remarks on result:
other: mean measured conc. +/- 10 % for the four higher conc. and +/- 20 % of nominal value for the lower conc.
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Remarks on result:
other: mean measured conc. +/- 10 % for the four higher conc. and +/- 20 % of nominal value for the lower conc.

It proved difficult to keep the concentrations of the test substance  constant throughout the test due to the fast dissipation from the water  body (rem. by reviewer: MAA is very rapidly biodegradable). Thus, some  analytical results show variability being higher than recommended in the  guideline. In the first test segment, the range of ± 20 % of the mean  measured values was exceeded by the standard deviations of the two lowest  concentrations. Regarding the individual values, the two lowest  concentrations were measured with more than half of the values outside  the range of ± 20 %, at 10 and 23 mg/l 8 % to 33 % of the measured  results were outside that range, respectively, while at the highest  concentration all measurements were within that range. Nevertheless,  there was no overlap of ranges of different concentrations: Each  individual value of each concentration was higher than the highest value  of the next lower concentration and lower than the lowest value of the  next higher concentration. 

Measured concentrations (2 mg/l and 51 mg/l not shown)

Segment1 --4.5 mg/l--  --10 mg/l -- --23 mg/l --
Mean      4.13   4.12   10.2  10.3   20.8  21.2
% of   
nominal  91.8   91.6   102.0 103.0   90.4  92.2
s.dev.    0.96   0.97    1.48  1.83   2.96  2.79
%        23.5   23.6    14.5  17.8   14.3  13.2 
Min       2.56   2.48    7.86  7.90  15.1  14.9 
Max       5.48   5.52   12.9  13.9   23.8  24.6 
Values    7      7       2     4      2     1 
out of
+20%     58 %   58 %    17 %  33 %   17 %   8 %
 

Segment2 --4.5 mg/l--  --10 mg/l -- --23 mg/l --
Mean      4.98   4.88   10.5  10.6   22.6  22.9
% of  
nominal 110.7  108.4   105.3 106.0   98.4  99.8 
s.dev.    0.71   0.92    1.46  1.24   1.82  1.83
%        14.3   18.9    13.8  11.7    8.0   8.0 
Min       3.64   3.45    8.08  8.67  18.5  19.8 
Max       5.76   6.32   12.6  12.5   25.2  25.6 
Values    2      4       1     0      0     0 
out of 
+ 20%    17 %   33 %     8 %   0 %    0 %   0 %

During the crucial, sensitive phase of the study (early stage of  free-feeding larvae, days 10-21) only one value in segment 1 was out of  the ± 20 % range and none in segment 2.


Effect data:

Hatch: By day 3 hatch success was (qualitatively) higher than 50 %, up to  day 5 hatching was complete. No statistically significant difference was  observed with regard to hatch success; on day 6 all replicates showed a  hatch success of 88 % or higher with the exception of one replicate at  the highest dose (60 %).

Survival: The trends regarding survival rates throughout the study can be  divided into three phases:
a) embryo and sac fry stage approx. up to day 10; survival in general >=  80 %,
b) free feeding larvae up to day 21; substantial differences concerning  mortality,
c) juvenile fish until study termination; low additional mortality in all  vessels.

LC50 and LC10-values from probit analyses of total fish counts (mean  survival for each concentration)

day   Mean control    LC50    LC10    C.L. of LC50
      survival (%)   (mg/l)  (mg/l)      (mg/l)
---------------------------------------------------
14        83.8         68      24       58 - 81
21        75.6         43      17       37 - 50
28        71.4         41      17       35 - 47
35        71.2         42      10       35 - 52
---------------------------------------------------

In order to determine a NOEC-value concerning the endpoint "survival",  the survival rates at study termination were used. Concerning post hatch  success a NOEC of 10 mg/l and an LOEC of 23 mg/l could be observed. 

In the following tables the first line represents segment 1 data and the  second line segment 2.

Survival rates (%) at study termination and results 
of ANOVA followed by Williams' test        
(data for 2 mg/l not shown)

Overall Survival (replicates and mean)
Controls | 4.5 mg/l| 10 mg/l | 23 mg/l | 51 mg/l
---------------------------------------------------
67.7 73.8|73.1 61.2|31.1 60.4|37.3 64.4|41.2 17.8
73.3 70.0|71.3 74.5|69.3 71.6|59.4 63.0|
  71.2   |  70.0   |  58.1   |  56.0   |  29.5 *
---------------------------------------------------
Post-Hatch Survival (replicates and mean)
Controls | 4.5 mg/l| 10 mg/l | 23 mg/l | 51 mg/l
---------------------------------------------------
74.2 80.9|76.8 64.3|32.7 68.5|41.8 69.1|41.6 29.5
77.1 72.9|72.7 80.0|74.5 72.3|63.2 67.0|
  76.3   |  73.5   |  62.0   |  60.3 * |  35.6 *
---------------------------------------------------
* significant difference from control at p   0.05


Weight/fish (% of control) at study termination
(data for 2 and 4.5 mg/l not shown)

Controls  |  10 mg/l |  23 mg/l  | 51 mg/l
---------------------------------------------------
107.7 92.3|86.4  90.5| 57.2  64.5|60.1 62.3
105.4 94.6|86.6 118.4|116.2 122.7|
---------------------------------------------------
Length/fish at study termination in cm
(data for 2 mg/l not shown)
Controls | 4.5 mg/l| 10 mg/l | 23 mg/l | 51 mg/l
---------------------------------------------------
1.30 1.29|1.33 1.27|1.31 1.25|1.17 1.27|1.13 1.09
1.24 1.23|1.18 1.19|1.19 1.21|1.22 1.20|
---------------------------------------------------

At the end of the study, individual lengths (digital image processing)  and group weights were determined. In test segments 1 and 2, different  trends could be observed: In test segment 1 a clear reduction of weight  and, less pronounced, length at 23 and 51 mg/l was evident, in test  segment 2 the groups at 23 mg/l showed the highest individual weight and  no difference to control lengths. The evaluation of combined data does  not reveal additional findings due to the high variance and missing  normal distribution at that concentration.

Other effects:
No other observations have been reported.

Validity criteria fulfilled:
yes
Conclusions:
Methacrylic anhydride rapidly hydrolysis to methacrylic acid in aqueous media. Therefore assessment of aquatic toxicity to Methacrylic anhydride based on studies with methacrylic acid is recommended.
No statistically significant effect on hatching success could be observed at any concentration tested. MAA impaired survival mainly at the life stage of free feeding larvae; post hatch success was decreased significantly at 23 mg/l and higher concentrations. At the highest concentration of 51 mg/l, also growth was reduced significantly. Thus, after 35 days (study termination) the NOEC was observed with 10 mg/l, the LOEC with 23 mg/l. No statistically significant effect on hatching success could be observed at any concentration tested. MAA impaired survival mainly at the life stage of free feeding larvae; post hatch success was decreased significantly at 23 mg/l and higher concentrations. At the highest concentration of 51 mg/l, also growth was reduced significantly. Thus, after 35 days (study termination) the NOEC was observed with 10 mg/l, the LOEC with 23 mg/l.
Executive summary:

Methacrylic anhydride rapidly hydrolysis to methacrylic acid in aqueous media. Therefore assessment of aquatic toxicity to Methacrylic anhydride based on studies with methacrylic acid is recommended.

Methacrylic acid was tested in Zebra Fish (Danio rerio), Early Life Stage Toxicity Test (OECD 210) at nominal test concentrations of 2.0, 4.5, 10, 23 and 51 mg/l for 35 days. No statistically significant effect on hatching success could be observed at any concentration tested. MAA impaired survival mainly at the life stage of free feeding larvae; post hatch success was decreased significantly at 23 mg/l and higher concentrations. At the highest concentration of 51 mg/l, also growth was reduced significantly. Thus, after 35 days (study termination) the NOEC and EC 10 was observed with 10 mg/l, the LOEC with 23 mg/l. No statistically significant effect on hatching success could be observed at any concentration tested. MAA impaired survival mainly at the life stage of free feeding larvae; post hatch success was decreased significantly at 23 mg/l and higher concentrations. At the highest concentration of 51 mg/l, also growth was reduced significantly. Thus, after 35 days (study termination) the NOEC and EC10 was observed with 10 mg/l, the LOEC with 23 mg/l.

Description of key information

NOEC (35d) for freshwater fish of the hydrolysis product methacrylic acid: 10 mg/l 

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
10 mg/L

Additional information