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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Nov 2011 to 09 Dez 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study OECD 211, GLP, analytical monitoring.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
adopted October 03, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Official Journal of the European Union (EN), dated May 30, 2008
Deviations:
no
Principles of method if other than guideline:
A semi-static test design (water renewal three times per week Monday, Wednesday and Friday) in closed bott!es without headspace was chosen because of the vo!atility of the test item.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: STRAUS (Clone 5)
- Source: IBACON laboratories, Germany
- Age at study initiation (mean and range, SD): 1.75 to 18 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 The cultivation of the parental Daphnia was performed in reconstituted water of a similar quality regarding to pH, components of the main ions and total hardness as the test water used in the test. The test organisms were not first brood progeny. The
Daphnia in the stock culture were fed at least all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of
IBACON.
- Food type:unicelluar green algae Desmodesmus subspicatus
- Amount: 0.1 to 0.2 mg C/Daphnia/day
- Frequency: daily, all working days

ACCLIMATION
- Acclimation period: not necessary, test organism is bred at test facility, breeding is performed in the same kind of test water
Test type:
semi-static
Limit test:
no
Total exposure duration:
21 d
Test temperature:
19 - 21 °C ±1°C C in the freshly prepared media;
19 - 20 °C in the aged media;
pH:
pH-value: 7.4 to 8.7; should not vary by more than 1.5 units
Dissolved oxygen:
7.8 to 10.7 mg/L in the aged media
Nominal and measured concentrations:
control (test water), 0.32, 1.0, 3.2, 10 and 32 mg/L nominal corresponding to 0.158, 0.49, 1.58, 5.05 and 23.1 mg/L geometric mean measured concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): closed because of volatile test substance
- Material, size, headspace, fill volume: 4.5 (ID) x 9.5 (H) cm, ca. 130 mL; filled up with the test solutions having nearly no headspace to reduce losses of the test item
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): test concentrations and the control were renewed on Days 3, 5, 7, 10, 12, 14, 17 and 19 of the
exposure period
- 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 250 mg CaCO3/L; Alkalinity: 0.9 mmol/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: 860 - 1090 lux (not exceeding 1500 lux) The light intensity was measured once during the test.

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 recorded
once 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.:
> 5.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Confidence interval, p=95 % was not determined
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
7.22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Confidence interval, p=95 % was not determined
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
23.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
32.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Results:

Survival of Adults: In the control and the test item concentrations of 1 and 3.2 mg test item/L, all Daphnia survived until the end of the test. At the
nominal concentrations of 0.32 and 10 mg test item/L 1 animal died during the test. At the highest test concentration of nominal 32 mg test item/L,
all Daphnia died during the course of the test. Thus, the survival rate of Daphnia magna after 21 days was first significantly reduced first at the
nominal test concentration of 32 mg test item/L.

Reproduction: The reproduction of Daphnia in the control was 114 ± 27 (mean ± SD) live offspring per adult. The coefficient of variation around
the mean number of living offspring produced per parent animal in the control was 23.5 %. According to the results of a Williams-test (one-sided,
α= 0.05) no significant toxic effect of the test item on the mean reproduction was determined up to and including the nominal test concentration of 10 mg test item/L. At the nominal concentration of 32 mg test item/L, all daphnia were dead at the end of the test and no reproduction was observed during the test. The EC50 of the reproduction was determined to be > 5.05 mg test item/L.

Date of First Brood: The first young Daphnia released from their parent animals were recorded in the control and the nominal test concentrations of 0.32, 1 and 10 mg test item/L at the observation on day 8. At the nominal concentration of 3.2 mg test item/L, the first offspring was recorded on day 9. At the highest concentration tested (32 mg test item/l), no offspring was produced.

Signs of Intoxication: With exception of the reported mortality and the reduced reproduction, no particular signs of intoxication were observed
during the test.

Appearance of the Test Item in the Test Media: No remarkable observations.
Reported statistics and error estimates:
The NOEC and the LOEC for the reproduction and survival were evaluated by the Williams Multiple Sequential t-test, Fisher’s exact and Welch t-test,
respectively. The EC50, EC20 and EC10 of the reproduction were determined by probit analysis.
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Analytical Results

Determination of the Test Item: Based on the results of HPLC measurements the concentration of the test item was determined using a calibration curve. The calibration curve was splitted as the linearity of the lower concentration range was influenced by the higher calibration standards.
Calibration Range: Low calibration range: 0.25 to 5 mg test item/L
High calibration range: 2.5 to 20 mg test item/L
Linearity of Response: Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression
Regression Coefficient (r2): Low calibration: at least 0.9979
High calibration: at least 0.9980
Typical Calibration Curve: Low calibration: y = 168775 * x + 8451
High calibration: y = 424147 * x + 248652
Limit of Detection: 0.078mg test item/L
Limit of Quantification: 1 mg test item/L96 % (n = 4, RSD 3 %)
Mean Recovery in the Fortified Samples: 100 % (n = 8, RSD 11 %)
Mean Recovery in the Test Samples: freshly prepared: 86 % (n = 18, SD 7 %)aged:                  45% (n = 12, SD 44 %)

Summary of Analytical Results

sample [mg/L]

geometric mean

 
 n
 control  n.a.  6
 3.2  49  6
 10  51  6
 32  72  6

1mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable
In the freshly prepared test media 86 % of the nominal test concentrations were found (average from all test concentrations equal and higher than the NOEC).A decrease of test item concentration was determined during the test medium renewal periods of 48 and 72 hours. Therefore, the biological results are related to geometric mean values.
Validity criteria fulfilled:
yes
Remarks:
To prove the validity of the test system and test conditions, an acute immobilization test according to DIN 38412 L 11 is carried out with potassium dichromate as reference item once per month.
Conclusions:
In this valid guideline study (OECD 211, semi-static conditions), the 21 day reproductive toxicity to daphnids was determined. Furthermore the
influence of Ethyleneglycol dimethacrylate on the survival of Daphnia magna was determined. The EC50 (reproduction) for Daphnia magna (STRAUS Clone 5) was > 5.05 mg/L, NOEC (reproduction and survival): 5.05 mg/L and LOEC (reproduction): 23.1 mg/L for Ethyleneglycol dimethacrylate.
All effect values given are based on the geometric mean measured test concentrations of Ethyleneglycol dimethacrylate.
Executive summary:

The 21-day-chronic toxicity of to Daphnia magna STRAUS (Clone 5) was studied under semi-static conditions. Daphnids were exposed to control, and test chemical at measured concentrations of 0.158, 0.49, 1.58, 5.05 and 23.1 mg/L (geometric mean). The 21 day EC50based on reproductive effects was > 5.05 mg a.i./L. The 21-day NOEC based on reproductive effects was 5.05 mg a.i./L (the highest test concentration with no significant reduction of the reproductive output). There was no reproductive output at the highest concentration level.

The first brood was released till day 8 to 9 by all parent animals of the control and the concentration levels except the highest test concentration.

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

 

Results Synopsis

 

Test Organism Age: Daphnia magna, 1.75 hr to 18 hr old

Test Type: semi-static, closed system

 

NOEC(reproduction): 5.05 mg a.i./L

LOEC(reproduction): 23.1 mg a.i./L                     Endpoint(s) Effected: reproductive output, IR 

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

One long-term study in freshwater invertebrates is available for Ethyleneglycol dimethacrylate.

The long term toxicity of the substance to Daphnia magna has been evaluated in a 21 day semi-static reproductive toxicity test according to OECD guideline 211 and EU method C.20.

Daphnids were exposed to control and test chemical at measured concentrations of 0.32, 1.0, 3.2, 10 and 32 mg/L nominal corresponding to 0.158, 0.49, 1.58, 5.05 and 23.1 mg/L geometric mean measured concentrations.

The 21-day EC10 was found to be 7.22 mg/l (based on reproduction) and the 21-day NOEC was 5.05 mg/l (based on reproduction and mortality).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
7.22 mg/L

Additional information