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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restricitions, GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on test solutions:
Because of its low solubility, the preparation of the highest test item  concentration was performed according to the CONCAWE test protocol and to the ASTM standard D6081-97 (WAF method). The subsequent test concentrations were prepared by serial dilution of the highest achievable concentration (WAF). The turbidity of the WAF was measured in order to detect undissolved test material. Only on day 3slight turbidity of the WAF was detected.

Dilution water: Purified drinking water was be used. The purification included filtration with activated charcoal, passage through a lime-stone  column and aeration until oxygen saturation. Carbonate hardness of the  water is approx. 90 mg/L CaCO3, pH is in the range of 7.5-8.5.  Conductivity, total hardness, alkalinity, as well as nitrate, nitrite,  ammonium, phosphate, and DOC contents of the dilution water are measured  regularly.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test Organisms:  
a) Age: 4 - 24 hours old  
b) Supplier/Source: Umweltbundesamt (German Federal Environment Agency),  Institut für Wasser-, Boden- und Lufthygiene, bred in the laboratory of  the Fh-IME (testing facility)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
20 - 20.1 °C
pH:
7.5 - 9.56 (see remarks)
Dissolved oxygen:
 3.0 - 8.9 mg/L (34 - 100 %) (see remarks)
Details on test conditions:
- Test Conditions: In accordance with the OECD Guideline; the test organisms were exposed to  five concentrations of the test item for a period of 21 days. The test  solutions were filled into the test vessels, the daphnids were added and  the vessels were sealed with a gas-tight stopper. On days 0, 3, 5, 7, 10  and afterwards every second day the water was renewed by transferring the  daphnids to new vessels with freshly prepared test solutions. For each test concentration and for the control 10x1 animals were used.  Each daphnid has been exposed separately in a completely filled and  numbered vessel containing 250 mL of test medium.

General test conditions The animals were fed at each renewal with suspensions of the unicellular  alga Scenedesmus subspicatus. The content of food in the test suspensions  (measured as turbidity at 758 nm) increased during the test from 7 mg C/L  equivalents to 15 mg C/L equivalents. pH value, oxygen concentration, and  temperature were checked directly before adding the animals, and before  and after each renewal. The vessels were closed with gas-tight stoppers  and subjected to a light/dark cycle of 16/8 hours. The temperature during  the test was within a range of 20 - 20.1 °C, and the light intensity was  712 - 774 lux.

- Statistical Method: For immobilization and reproduction the NOEC, LOEC, and the EC50 were  determined: A LOEC has been calculated using ANOVA followed by Williams'  test. The data were analyzed by regression to determine the EC50  including the 95% confidence interval as well as the  EC10 using  Probit-analysis assuming log-normal distribution of the values.
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.105 mg/L
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.219 mg/L
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.2 mg/L
Basis for effect:
reproduction
Remarks on result:
other: (95 % C.I.; 0.19 - 0.22 mg/L)

The following endpoints observed in the reproduction test were evaluated quantitatively: Mortality (immobility) of parental generation daphnids; time to the first brood; number of live offspring per surviving female; intrinsic rate of increase r; individual length of adults

- Measured Test Concentrations: 
------------------------------------------------------
                       Measured Concentration, mg/L
                  ------------------------------------
          0d    0d     3d     3d      5d      5d     7d
         new   old    new    old     new     old    new
------------------------------------------------------

Control  0.010    *   0.100  <LOQ   0.010    <LOQ   0.020
1        0.232    *   0.243  0.098  0.010    <LOQ   0.020
2        0.303    *   0.523  0.236  0.245    0.220  0.320
3        0.585    *   0.956  0.455  0.522    0.650  0.682
4        1.204    *   1.713  0.884  1.122    0.921  1.138
5        2.763    *   4.123  2.009     *     2.398     *

-----------------------------------------------------
                      Measured Conc., mg/L     
               --------------------------------------
                7d     10d    10d    12d    12d     
               old     new    old    new    old            
-----------------------------------------------------
Control       0.020   0.010   0.010  <LOQ   <LOQ
1             0.053   0.130   0.051  0.180  0.040
2             0.124   0.267   0.071  0.330  0.090
3                *       *       *      *      * 
4                *       *       *      *      *
5                *       *       *      *      *
-----------------------------------------------------

                      Measured Conc., mg/L     
               --------------------------------------
               14d     14d    16d    16d    18d  
               new     old    new    old    new      
-----------------------------------------------------
Control       0.010   0.008  <LOQ    0.015  <LOQ
1             0.190   0.070  0.220   0.050  0.150    
2             0.420   0.120  0.430   0.120  0.350 
3                *       *       *      *      * 
4                *       *       *      *      *
5                *       *       *      *      *
-----------------------------------------------------

                      Measured Conc., mg/L     
               -----------------------------------
               18d     20d    20d    21d   21d   |  
               old     new    old    new   old   |  
--------------------------------------------------
Control        <LOQ    <LOQ   <LOQ    *   <LOQ   | 
1              0.030   0.160  0.040   *   0.090  | 
2              0.080   0.310  0.160   *   0.210  | 
3                 *       *     *     *     *    |  
4                 *       *     *     *     *    | 
5                 *       *     *     *     *    |  
--------------------------------------------------

5 = Water accommodated fraction (WAF, max. achievable conc.); 4 = WAF  1:2; 3 = WAF 1:4; 2 = WAF 1:8; 1 = WAF 1:16; all dilutions with dilution  water

new: freshly prepared test solution
old: old test solution before renewal

* not sampled
LOQ: limit of quantification at 0.0075 mg/L


Test substance concentration (mg/l) - arithmetic means of geometric means  for each test interval
----------------------------------------------------------
interval     WAF        Control°   Conc 1    Conc 2
 (day)    (initial)
----------------------------------------------------------
 0 to 3      2.76        0.005     0.151      0.267
 3 to 5      4.12        0.006     0.157      0.339
 5 to 7      2.58        0.014     0.086      0.174
 7 to 10     n.m         0.014     0.092      0.151
10 to 12     2.57        0.004     0.072      0.155
12 to 14     3.07        0.001     0.112      0.199
14 to 16     3.44        0.012     0.097      0.224
16 to 18     3.51        0.000     0.081      0.185
18 to 20     2.32        0.000     0.077      0.237
20 to 21     2.44        0.000     0.120      0.255
----------------------------------------------------------
 mean        2.98        0.006     0.105      0.219      


----------------------------------------------------------
interval    Conc 3       Conc 4    Conc 5
 (day)    
----------------------------------------------------------
 0 to 3      0.516       1.032     2.356
 3 to 5      0.788       1.256     3.144
 5 to 7      0.342       0.778       *
10 to 12     0.294       0.817       *     
12 to 14       *           *         *        
14 to 16       *           *         *        
16 to 18       *           *         *        
18 to 20       *           *         *    
---------------------------------------------------------- mean         0.485       0.971     2.750
20 to 21       *           *         *        
----------------------------------------------------------
 mean        2.98        0.006     0.105      0.219      

°Remark: In half of the control samples concentrations slightly above the  LOQ were found, corresponding to less than 10% of the concentrations  measured for the NOEC. The findings were most probably due to glassware  contamination. These findings have no influence on the validity of the  test.

- Water chemistry 

- pH: 
High initial pH-values were found particularly at 0.971 mg/l (up to pH  9.1) and 2.750 mg/l(up to pH 9.6). It is assumed that this is due to the  loss of carbon dioxide from the test medium during sterilization and  re-oxygenation of the dilution water for the WAF procedure. All pH values  were still within the physiological tolerance limits of the test species  and the key effects of the test occurred at concentrations without  significant pH shift (see table below). Therefore, it is assumed that pH  did not have an influence on the test results. 
-----------------------------------------------------
pH      CONTROL        0.105mg/l       0.219 mg/l  
DAY   new     old     new     old     new     old
        Medium          Medium          Medium
-----------------------------------------------------
0    8.11            8.22            8.30    
3    7.97    8.22    8.00    8.31    8.05    8.35
5    7.89    8.03    7.93    8.05    8.02    8.10
7    8.09    8.13    8.23    8.12    8.35    8.01
10   8.11    8.01    8.19    7.81    8.30    7.63
12   7.89    8.00    8.04    7.82    8.26    7.74
14   8.10    8.03    8.08    8.34    8.28    8.37
16   8.07    8.11    8.17    8.53    8.28    8.47
18   8.08    8.34    8.13    8.34    8.34    8.42
20   8.00    8.23    8.04    8.50    8.17    8.55
21           8.14            8.15            8.18
-----------------------------------------------------

- Water chemistry - dissolved oxygen (DO)
DO:
Low oxygen saturation in old medium before renewal was related to test  item concentrations and most likely attributable to biodegradation.  Oxygen levels decreased especially from day 7 to 10, when the period  between test medium exchanges was 3 days. Therefore, test media were  changed every 2 days afterwards.
-----------------------------------------------------
O2 %  CONTROL    0.105 mg/l   0.219 mg/l   0.485 mg/l  
DAY   new old     new old     new old     new old
       Medium      Medium      Medium      Medium
-----------------------------------------------------
0     97          97         100          95     
3     97   95     97   96     98   95     98   95
5     95   89    100   89     99   88     99   87
7     97   85     98   80     96   78     96   75
10    99   74    100   72     99   42          34
12   100   80    100   62    100   42
14    99   74     99   85     98   87
16   100   81     98   89     98   88
18    98   88     99   84     96   84
20    98   88     98   86     95   86
21         96          93          90
-----------------------------------------------------

Mortality

- Cumulative Number of Dead Parental Daphnids
 After 9 days of exposure, mortalities of 0, 80, and 100 % were observed  among daphnids exposed to the treatment levels 1, 2, and 3 - 5,  respectively. All daphnids died by the 5th day at conc. 5 ( mean: 2.750  mg/l). All daphnids survived to the end of the test period at conc. 1  (mean: 0.105 mg/l).

------------------------------------------------------
      Cumulative Mortality of Parental Daphnids [%]
Conc.      Days  1   2   3   4   5   6   7   8   9  10
------------------------------------------------------
Control          0   0   0   0   0   0   0   0   0   0
0.105 mg/l        0   0   0   0   0   0   0   0   0   0
0.219 mg/l        0   0   0   0   0   0   0   0  80  80
0.485 mg/l        0   0   0   0   0   0   0   0 100 100
0.972 mg/l        0   0  20  20  20  20  20  20 100 100
2.750 mg/l        0   0  80  80 100 100 100 100 100 100
------------------------------------------------------
      Cumulative Mortality of Parental Daphnids [%]
Conc.  Days 11  12  13  14  15  16  17  18  19  20  21
------------------------------------------------------
Control      0   0   0   0   0   0   0   0   0   1   1
0.105 mg/l    0   0   0   0   0   0   0   0   0   0   0
0.219 mg/l   80  80  80  80  80  80  80  80  80  80  80
0.485 mg/l  100 100 100 100 100 100 100 100 100 100 100
0.971 mg/l  100 100 100 100 100 100 100 100 100 100 100
2.750 mg/l  100 100 100 100 100 100 100 100 100 100 100 
------------------------------------------------------
Effect Data (mortality(immobilization)

Endpoint Immobility/Survival
EC10         0.154
EC50         0.191
LOEC         0.219
NOEC  0.105

------------------------------------------------------
Reproduction - Time of First Brood, No. of Offspring 

All newborn daphnids of dead maternal daphnids were also dead. One of the  two females at 0.219 mg/l had a total of 20 surviving and 7 dead  newborns. No reproduction occurred at 0.53 mg/l and 1.01 mg/l; there were  no surviving maternal daphnids at 2.77 mg/l.
------------------------------------------------------
Reproduction        control   0.105 mg/l  0.219 mg/l
------------------------------------------------------
Time to first brood    8.4 d     8.7 d      11 d

After day 9 (%)        0         0          50

Mean offsp. /female   74.1      69.9        29.5***

Standard deviation     8.6      11.7        13.4

Relative s.d.         11.6 %    16.7 %      45.5 %   
------------------------------------------------------
Statistical evaluation by one-sided t-tests:
* p < 0.05;  ** p < 0.01:  *** p < 0.001

-Effect Data (reproduction)
 EC50 (21day) = 0.20 mg/L (95 % C.I.; 0.19 - 0.22 mg/L) 
 EC10 (21day) = 0.14 mg/L 
 NOEC = 0.105 mg/L
 LOEC = 0.219 mg/l


Size Measurements at Study Termination
------------------------------------------------------
Length of Maternal Daphnids After 21 Days
                    control    0.105 mg/l   0.219 mg/l
------------------------------------------------------
mean length (mm)      4.54       4.26*       3.87**
standard deviation    0.31       0.17        0.20
relative s.d.         6.8%       4.0%        5.2%
statistical evaluation by one-sided t-tests: 
* p< 0.05, ** p< 0.01, *** p< 0.001

No NOEC can be determined statistically. An analysis of the reproductive  success vs. body size between the maternal daphnids of the control and  the lowest concentration, 0.105 mg/l, indicated that there is no observable correlation between body size and reproduction. Therefore, the difference in size is regarded as ecotoxicologically unimportant.


Conclusions

The test item concentrations (arithmetric means of measured values)  differed by a factor of about 2, as intended.

In spite of the difficult study conditions, the test was valid regarding the controls: Water quality, mortality, timing and amount of reproduction  met the quality criteria of OECD 211. Also the lowest concentration met  the OECD criteria for controls. 

There was total mortality of maternal daphnids at the three highest  concentrations. Concentration 2 also exhibited high mortality. Mortality  was correlated with low oxygen levels. It remains unclear whether those  were contributing to the mortality or were a result of it. In any case, mortality can be attributed to the test item concentration.

Concentration 2 exhibited also clear effect on reproduction and growth.

The NOEC based on three population-relevant parameters mortality,  generation time and reproduction is consistently determined at 0.105  mg/L. In all cases it is below the EC10 as calculated threshold value.  This is due to the fact that the variability between the 10 replicates  was low, resulting in a high statistical power.

For the same reason, length measurement results were statistically  significantly different between controls and the lowest concentration.  However, the small difference of 4.3 compared with 4.5 mm and the fact  that there were no effects on reproduction are considered as arguments  for considering the NOEC of <0.105 mg/L as irrelevant for the evaluation  of the study.

Conclusions:
In a valid guideline study, the 21 d NOEC of the test substance to reproduction of Daphnia magna was 0.105 mg/L.
Executive summary:

In a valid guideline study, the 21 d NOEC of the test substance to reproduction of Daphnia magna was 0.105 mg/L. In addition, information on acute toxicity to mother daphnia can be used. No toxicity is observed at 24 and 48 hours but at 72h 80% mortality is observed in the highest concentration 2.75 mg/L and 20% mortality at 0.97 mg/L. It can can concluded, based on these results, that acute toxicity is in the range of limit of solubility.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-11 to 2010-08-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study OECD 211, GLP, analytical monitoring.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Germany
- Age at study initiation (mean and range, SD): 2 to 24 hours
- Length at study initiation (length definition, mean, range and SD): no data
- Weight at study initiation (mean and range, SD): mean weight: no data
- Method of breeding: Laboratory bred
- Food type: unicellular green algae e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Cholorella vulgaris
- Amount: corresponding to 0.2 mg C per daphnia and day
- Frequency: daily

ACCLIMATION
- Acclimation period: test organism is bred at test facility
- Health during acclimation (any mortality observed): no data
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
Total hardness: at test concentrations: (118 µg/L Isodecyl methacrylate): 152 - 182 mg/L as CaCO3
control: 162 to 182 mg/L as CaCO3
Test temperature:
20.8 °C - 22 °C
pH:
at test concentrations: 6.28 to 7.82
control: 7.31 to 8.04
Dissolved oxygen:
at test concentrations: 5.27 to 8.79 mg/L
control: 7.56 to 8.92 mg/L
Nominal and measured concentrations:
1:1 (saturated test item solution), 1:2, 1:4, 1:8, 1:16 prepared with dilution water
corresponding to geometric mean measured concentrations: 118, 54.2, 27.9, 13.7, 6.38 µg/L
Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers, 100 mL capacity
-Test volume: 50 mL
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Total organic carbon: 160 to 180 mg CaCO3/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen concentration, pH-values, total hardness and water temperature were measured once
every 7 days, in fresh and old media, in one replicate of the control and the saturated solution or the next appropriate dilution level.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hr light / 8 hr dark cycle
- Light intensity: max. 20 µE*mE-2*sE-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Mortality of parental daphnids
The number of parental daphnids which were immobilized or dying during the test and day of death were observed and recorded once a day at
least at the same time as the offspring was counted. Dead specimen were removed.

Neonates
First appearance of juveniles was checked daily. The number of neonates (alive and dead progeny) was counted and dates of release were recordedonce a day from the first day of production of neonates unti! the end of the test. The neonates (alive and dead) were removed after counting and
before addition of algae to prevent from consuming food intended for the adults. The number of aborted eggs or dead offspring and dates of
observation were recorded.

Condition of parental daphnids
Abnormalities (e.g. swimming behaviour, number of male and winter eggs) were observed and recorded at each day of observation.

Total length and dry body weight of parental daphnids
At the end of the test total length of each daphnid and the mean dry body weight of the daphnids alive in all dilution levels and control were
determined.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 118 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
118 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
54.2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
118 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
adult daphnids
Remarks on result:
other: No mortality was observed at the highest test concentration.
Details on results:
- Mortality of parent animals: The test item induced no adult mortality levels at any test concentration after 21 days.
- Body length and weight of parent animals: The mean values of the body length of the surviving parental daphnids at the test groups were in the
range of 4.65 to 4.95 mm per daphnid were comparable to the control group with 4.70 mm per daphnid. The mean dry body weight was determined to be in the range of 0.57 to 0.84 mg per daphnid at the test groups and 0.67 mg per daphnid at the control, respectively.
- Time to first brood release or time to hatch: The first brood was released till day 9 by all parent animals of the control and the concentration levels 6.38 to 27.9 µg/L.
7 of 10 parental daphnids of the concentration level 118µg/L released their first brood significantly delayed on day 10 or 11.
Release of the first brood after day 9 is regarded to be too late and biologically significant.
Results with reference substance (positive control):
- Results with reference substance valid?: yes
_Reference Item: Potassium dichromate
- Relevant effect levels: EC50: 1.70 mg/L (CL: 1.51 - 1.97 mg/L), immobilization after 24 hr.
Reported statistics and error estimates:
One Way Analysis of Variance on Ranks, Dunnett's method, p=0.05

Test Item Related Effects on Reproduction (NOEC, LOEC, EC50)

 

Effects

Isodecyl methacrylate

Geometric mean measured test Item Concentration

[μg/L]

 

Control

6.32

13.7

27.9

54.2

118

Mean Number of Juveniles per Producing

Parent (Reproduction Rate ± SD)

 

122±9

118±11

115±8

122±8

115±10

74.1±201)

Coefficient of Variation of the Mean Number

of Juveniles per Producing Parent [%]

 

7

9

7

7

9

272)

Mean Intrinsic Rates of Natural Increase

0.50

0.50

0.50

0.50

0.50

0.401)

NOEC,Reproduction

54.2μg/L

LOEC,Reproduction

118μg/L

EC50,Reproduction

> 118μg/L

SD = Standard deviation

1)= Statistical significance (One Way Analysis of Variance, DUNNETTSmethod, p = 0.05)

2)= Coefficient of variation > 25 % is regarded to be biologically significant

Test Item Related Effects on Adult Mortality and Growth

 

Effects

Isodecyl methacrylate

Geometric mean measured test Item Concentration

[μg/L]

 

Control

6.32

13.7

27.9

54.2

118

Adult mortality after 21 days [%]

 

0

0

0

0

0

0

Parent animals: mean body length [mm]

 

4.70

4.65

4.73

4.95

4.78

4.65

Parent animals: mean dry weight [mg]

0.67

0.69

0.72

0.84

0.66

0.57

 

Validity criteria fulfilled:
yes
Conclusions:
In this unrestrictedly valid guideline study (OECD 211, semi-static conditions), the 21 day reproductive toxicity to daphnids was determined to be as follows: EC50 for Daphnia magna (STRAUS Clone 5) was > 118 µg/L, NOEC: 54.2 µg/L and LOEC: 118 µg/L for Isodecyl methacrylate. All effect values given are based on the geometric mean measured test concentrations.
Executive summary:

The 21-day-chronic toxicity of Isodecyl methacrylate to Daphnia magna STRAUS (Clone 5) was studied under semi-static (3 times per week renewal) conditions. Daphnidswere exposed to control, and test chemical at geometric mean measured concentrations of 6.38 – 13.7 – 27.9 – 54.2 – 118 µg/L.

The 21 day EC50based on reproductive effects was > 118 µg/L.  The 21-day NOEC based on reproductive effects was 54.2 mg µ/L (the highest test concentration with no significant reduction of the reproductive output). The reduction of the reproductive output at the concentration level of 118 µg/L was statistically significant in comparison to the control group. Isodecyl methacrylate induced no adult mortality up to the highest test concentration. The EC50 value for adult mortality was estimated directly from the observation data to be > 118 µg/L. The intrinsic rates of natural increase of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by KRUSKAL-WALLIS One Way Analysis of Variance on Ranks, DUNNETT’S method (p = 0.05). There was a statistically significant difference at the tested concentration level 118μg/L when compared to the control. At the other concentration levels 6.38 to 54.2μg/L no statistically significant difference in comparison to the control was determined. Thefirst broodwas released till day 9 by all parent animals of the control and the

concentration levels 6.38 to 27.9μg/L. One parental daphnid of the concentration level

54.2μg/L released its first brood on day 10. At the concentration level 118μg/L seven

parental daphnids released their first brood biologically significantly delayed on day 10 or 11.

Release of the first brood after day 9 is regarded to be too late and biologically significant.

Four to five broods were observed at the concentration levels 6.38 to 27.9μg/L and control

during the course of the study. Three broods were observed by one parental daphnid of the

test concentration level 118 μg/L. No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead and alive) the percentage of dead juveniles was biologically not significant at the tested concentration levels.

The mean values of the body length of the surviving parental daphnids at the test groups in the range of 4.65 to 4.95 mm per daphnid were comparable to the control group with 4.70 mm per daphnid. The mean dry body weight was determined to be in the range of 0.57 to 0.84 mg per daphnid at the test groups and 0.67 mg per daphnid at the control, respectively.

This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

NOTE:Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-02-15 to 2005-03-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study OECD211, GLP. At the limit concentration of 10 µg/l (nominal), corresponding to a time weighted mean measured concentration of 5.73 µg/l, which is above the limit of water solubility (< 1 µg/l), but which had to be used for analytical reasons (because of the LoQ of 1 µg/l), no effects of dodecyl methacrylate on reproduction or immobilisation of the daphnids were found. Valid with restriction: test concentration above the limit of solubility.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
adopted September 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10.0 µg/L
- Sampling method: Samples (10.0 µg/L, control) were stabilised with acetonitrile (1:1) immediately after sampling. 25 mL of new media and 50 mL of old media were used for trace analysis.
- Sample storage conditions before analysis: room temperature, protected from light and moisture
- Expiry date: 2005-03-14
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10.0 mg test item/L was weighed out onto the cap (with Teflon layer) of a 1 L glass bottle, filled up with 1 L demineralised water and shaken at ca. 20 rpm at room temperature for at least 20 h (rotating shaker 3040, GFL) to get a homogenous emulsion. The renewal frequency of the stock
solution was every 3 to 4 days.
- Controls: 10 replicates without test item tested under the same conditions as the test group
- Chemical name of vehicle: dilution water Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Source: Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu); Breeder: Dr. U. Noack-Laboratorien, D-31157 Sarstedt, Germany
- Age of parental stock (mean and range, SD):
- Feeding during test
- Food type: suspension
- Amount: A suspension of Desmodesmus subspicatus (0.385 - 0.670 mL) and Chlorella vulgaris (0.31 0 - 0.825 mL) per test vessel
(corresponding to 0.1 - 0.2 mg C per daphnid and day). It varied according to the growth of the daphnids, but was the same for the control and
test groups on any particular day.
- Frequency: 5 x weekly
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
Total hardness: > 140 mg/L
Test temperature:
18 - 22 °C ± 2 °C constant
pH:
Initial: 7.93 - 8.14; Final: 7.72 - 8.28 at the limit concentration
Initial: 7.85 - 8.17; Final: 7.73 - 8.33 control
Dissolved oxygen:
Final oxygen concentration: >= 8.30 mg/L (limit concentration and control)
Nominal and measured concentrations:
Limit concentration of nominal 10 µg/L, corresponding to the time weighted mean measured concentration of 5.73 µg/L.
Details on test conditions:
TEST SYSTEM
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: max. 20 µE * mE-2 * sE-1


EFFECT PARAMETERS MEASURED :
Mortality of parent Daphnia
The number of parent animals which were immobilised or dead during the test and day of death were observed and recorded once a day.
Juveniles
First appearance of juveniles was checked daily. The number of juveniles (alive and dead progeny) was counted and dates of release were recorded
once a day from the first day of production of juveniles until the end of the test. After counting the juveniles were discarded. The number of aborted
eggs and dates of observation were recorded.
Condition of parent Daphnia
Abnormalities (e.g. swimming behaviour, number of males and winter eggs) were observed and recorded each day of observation.
Total length and body weight of parent Daphnia
At the end of the test total length of each daphnid and body weight of the daphnids alive at the limit concentration and control were determined.

VEHICLE CONTROL PERFORMED: yes


RANGE-FINDING STUDY (21 d, non-GLP)
- Test concentrations: 10 µg/L
- Results used to determine the conditions for the definitive study: No effect on reproduction capacity and mortality of the parent animals was
observed.
Based on the results of the preliminary test the definitive study was performed as a limit test with a limit concentration of 10.0 µg/L above the water
solubility level and > LOQ (1 µg/L).
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 5.73 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and immobilisation
Remarks on result:
other: test concentration above the water solubility
Details on results:
- Mortality of parent animals: No mortality of parental and control animals during the test period.
- No. of offspring produced per day per female: not applicable, due to production of offspring was done mainly on day 9, 12, 15/16 and 19 of the
test period.
Mean number of offspring alive produced per parent animal after 21 days: 85 ± 4.27 (control group); 82.2 ± 2.30 (test group)
- Body length and weight of parent animals: Total length: 4.50 - 5.00 mm (treated animals); 4.50 - 5.25 mm (control animals)
Dry weight (mean value): 1.15 mg (treated animals; 1.19 (control animals)
No significant effects were determined.
- Type and number of behavioural abnormalities: none (test group and control group)
- Time to first brood release or time to hatch: mean day: 9.0 (control group); 8.8 (test group)
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels:
In order to prove the validity of the test system and test conditions at the testing facility, an acute immobilisation test according to DIN 38412 L11
was carried out with potassium dichromate as reference item once per month. The EC50 of the reference item at 1.84 mg/L (1.74 - 1.95
(95% confidence interval)) after 24 hours was within the validity range of 1.0 to 2.5 mg/L according to DIN 38412 L30.
Reported statistics and error estimates:
The NOEC at the limit concentration for the reproduction was determined directly from the results.
Significant deviations concerning reproduction were determined in comparison to the control group using statistical standard procedures (One Way Analysis of Variance, p < 0.05 for reproduction rate and intrinsic rate of natural increase and KRUSKAL-WALLISS ANOVA on Ranks, p < 0.05 for the
day of release of first brood). Significant deviations concerning the body length were determined in comparison with the control group using
KRUSKAL-WALLISS ANOVA on Ranks, p < 0.05.

Number of Juveniles in the Control and Test Group after 21 Days

TWM

[µg/L]

Number of Juveniles in Replicate No. 1 2 3 4 5 6 7 8 9 10 Total No. No. of Parents alive Mean No. of Juveniles per Parent alive [MV ± SD] CV [%] Comparison versus Control Red.[%] Stat.
 5.73 84 82 82 79 86 80 85 83 81 80  822  10 82.2  ± 2.30  2.80  3.29 no
 Control 85 95 85 83 84 79 83 88 82 86  850   10 85.0  ± 4.27  5.02 --- ---

TWM = Time weighted mean measured concentration

MV ± SD = Mean value ± Standard deviation

CV = Coefficient of variation

Red. = Reduction of reproduction

Stat. = Statistical significance (ANOVA, p < 0.05)

no = Statistically not significant

Summary of all test item related effects on repropduction, NOEC based on time weighted mean measured concentration of 5.73 µg/l

Effect

 

Number of Juveniles (Reproduction Rate)1)

no

Day of Release of 1stBrood1)

no

Stillborn Juveniles and Aborted Eggs

no

Intrinsic Rates (IR)1)

no

Summary of all Test Item Related Effects on Reproduction

no

1)     = Statistical significance (one Way Analysis of Variance, p < 0.05)

No = no significant effect

Validity criteria fulfilled:
yes
Conclusions:
At the limit concentration of nominal 10 µg/L, corresponding to the time weighted mean measured concentration of 5.73 µg/L, above the water
solubility level and > LOQ (< 1 µg/L) no effects of Dodecyl methacrylate on reproduction and immobilisation of daphnids was determined.
Executive summary:

The 21-day-chronic toxicity of Dodecyl methacrylate to Daphnia magna STRAUS (Clone 5) was studied under semi static

conditions according to OECD guideline 211. Daphnids were exposed to Dodecyl methacrylate at limit concentration of 10 µg/L (nominal).

This concentration is higher than the solubility in water (< 1 µg/L) but has, nevertheless, been choosen with regard to the feasibility of

attaining appropriate and analysable test concentrations at 10 µg/L.

10 test organisms, individually held were used for the limit concentration and control. At test start they were 2 to 24 hours old. The test method was semi-static and test solutions were renewed daily.

Concentrations of Dodecyl methacrylate in the stock solution, limit concentration and control of fresh (0 h) and old (24 h) media were determined via HPLC. Samples were taken and analysed on days 0, 7, 16, 20 (fresh media) and on days 1, 8, 17, 21 (old media). The test item concentrations decreased within 24 h. All effect values are given based on the time weighted mean measured concentration for the limit concentration of 5.73 µg/L.

The average number of juveniles per parent in the control group was 85 after 21 days. The reproductive output at the limit concentration was not statistically significant reduced compared to the control (One Way Analysis of Variance, p < 0.05).

The coefficient of variation around the mean number of living offspring produced per parent in the control group was 5.02 % and shows very small variances between the control replicates.

No winter eggs, males, ephippia, stillborn juveniles and aborted eggs occurred in control or test groups.

The mean day of release of first brood was 9 in the control group and the limit concentration. There was no difference between the two groups. At the limit concentration and the control group 4 broods were released during the test period.

The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and offspring numbers were used for calculation of population growth. The mean IR of the surviving daphnids of the limit concentration was compared to the control by One Way Analysis of Variances (p < 0.05). There was no statistically significant difference. The intrinsic rate was comparable for the control and limit concentration.

The no observed effect concentration (NOEC) after 21 days based on reproduction capacity is the tested limit concentration of 5.73

µg/L. No statistically significant test item related effects were observed at the limit concentration when compared to the control

group. No irnmobilisation of parent animals occurred in the control or test group.

Water quality parameters as pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits.

In order to prove the validity of the test system and test conditions at the testing facility, an acute immobilisation test according to DIN 38412 L11 was carried out with potassium dichromate as reference item once per month. The EC50 of the reference item at

1.84 mg/L after 24 hours was within the validity range of 1.0 to 2.5 mg/L according to DIN 38412 L30.

The 21 day LC50/EC50based on reproduction/immobilisation was greater than 5.73 µg/L (mean measured concentration). The 21-day NOEC based on reproduction/immobilisation was 5.73 µg/L (mean measured concentration). Production of offsprings in the treated groups indicated that Dodecyl methacrylate did not have an effect on the reproduction at concentrations greater than 5.73 µg/L.

 

This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

 

Results Synopsis

 

Test Organism Age: 2 to 24 hours old at test start

Test Type: Semi-static (renewal daily)

 

NOEL:  5.73 µg/L (based on time weighted mean measured concentration)                 

Endpoint(s) Effected:  reproduction/immobilisation

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included as attachment. Please also see attached justification.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The constituents of the UVCB substance NUMA (Nonyl-Undecyl methacrylate) are structurally related mono alkyl methacrylate esters differing only in the respective alcoholic moieties. The main proportion of these esters are of the n-type the minor proportion of the iso type. Considering the small amount of iso-types and the negligible differences in (eco-) toxicological properties between the n- and iso types, in this assessment both types of one ester with one specific chain length are assessed together as a whole.
Further information is included as attachment. Please also see attached justification Chapter 1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Further information is included as attachment. Please also see attached justification Chapter 1.

3. ANALOGUE APPROACH JUSTIFICATION
Please see attached justification Chapter 1 and 3.

4. DATA MATRIX
Please see attached justification Chapter 1.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
54.2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
118 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 118 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
118 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
adult daphnid
Remarks on result:
other: No mortality was observed at the highest test concentration.
Conclusions:
In analogy to an valid guideline study (OECD 211, semi-static conditions) with i-DMA, the 21 day reproductive toxicity to daphnids was determined to be as follows: EC50 for Daphnia magna (STRAUS Clone 5) was > 118 µg/L, NOEC: 54.2 µg/L and LOEC: 118 µg/L for Isodecyl methacrylate. All effect values given are based on the geometric mean measured test concentrations.
Executive summary:

There are no test data available for most of the constituents and the main proportion of the constituents are in the critical range of C8 -C11 methacrylates for aquatic toxicity, therefore the available data are used for a worst case approach. Regarding chronic aquatic effects NUMA with its constituents is assessed in analogy to i-DMA which is the worst case for aquatic toxicity. Considering this very conservative approach, the read across and the assessment are regarded as acceptable with a medium level of confidence.

The 21-day-chronic toxicity of Isodecyl methacrylate to Daphnia magna STRAUS (Clone 5) was studied under semi-static (3 times per week renewal) conditions. Daphnidswere exposed to control, and test chemical at geometric mean measured concentrations of 6.38 – 13.7 – 27.9 – 54.2 – 118 µg/L.

The 21 day EC50based on reproductive effects was > 118 µg/L.  The 21-day NOEC based on reproductive effects was 54.2 mg µ/L (the highest test concentration with no significant reduction of the reproductive output). The reduction of the reproductive output at the concentration level of 118 µg/L was statistically significant in comparison to the control group. Isodecyl methacrylate induced no adult mortality up to the highest test concentration. The EC50 value for adult mortality was estimated directly from the observation data to be > 118 µg/L. The intrinsic rates of natural increase of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by KRUSKAL-WALLIS One Way Analysis of Variance on Ranks, DUNNETT’S method (p = 0.05). There was a statistically significant difference at the tested concentration level 118μg/L when compared to the control. At the other concentration levels 6.38 to 54.2μg/L no statistically significant difference in comparison to the control was determined. Thefirst broodwas released till day 9 by all parent animals of the control and the

concentration levels 6.38 to 27.9μg/L. One parental daphnid of the concentration level

54.2μg/L released its first brood on day 10. At the concentration level 118μg/L seven

parental daphnids released their first brood biologically significantly delayed on day 10 or 11.

Release of the first brood after day 9 is regarded to be too late and biologically significant.

Four to five broods were observed at the concentration levels 6.38 to 27.9μg/L and control

during the course of the study. Three broods were observed by one parental daphnid of the

test concentration level 118μg/L. No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead and alive) the percentage of dead juveniles was biologically not significant at the tested concentration levels.

The mean values of the body length of the surviving parental daphnids at the test groups in the range of 4.65 to 4.95 mm per daphnid were comparable to the control group with 4.70 mm per daphnid. The mean dry body weight was determined to be in the range of 0.57 to 0.84 mg per daphnid at the test groups and 0.67 mg per daphnid at the control, respectively.

This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

NOTE:Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Description of key information

There are no test data available for most of the constituents and the main proportion of the constituents are in the critical range of C8 -C11 methacrylates for aquatic toxicity, therefore the available data are used for a worst case approach. Regarding chronic aquatic effects NUMA with its constituents is assessed in analogy to i-DMA which is the worst case for aquatic toxicity.

The 21-day-chronic toxicity of Isodecyl methacrylate to Daphnia magna STRAUS (Clone 5) was studied under semi-static (3 times per week renewal) conditions. Daphnids were exposed to control, and test chemical at geometric mean measured concentrations of 6.38 – 13.7 – 27.9 – 54.2 – 118 µg/L.

The 21 day EC50based on reproductive effects was > 118 µg/L.  The 21-day NOEC based on reproductive effects was 54.2 mg µ/L (the highest test concentration with no significant reduction of the reproductive output).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
54.2 µg/L

Additional information