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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:
2009-06-09 to 2009-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
ANALYSIS OF THE TEST ITEM CONCENTRATIONS
For determination of the test item concentrations, duplicate samples were taken from the freshly prepared test media of all test concentrations and from the control at one test medium renewal period of the first, second and last week (Days 0, 7 and 16, respectively).

To determine the maintenance of the test item concentrations in the test media, stability samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19).

The last samples from the highest nominal concentration of 0.63 mg/L were taken on Day 9 as all daphnids were dead at observation on Day 12 at this concentration level.

The following stability samples were taken in duplicate:

a) Samples with food and test animals, taken from the actual test by combining the contents of all replicate test beakers after the end of the test medium renewal periods.
b) Samples without food and test animals, incubated during the renewal periods under the test conditions.

All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments (without GLP) for investigation of the storage stability, the test item was considered to be stable in the test water under these storage conditions.

The concentrations of the test item were measured in all duplicate test media samples (with or without food and test animals) from the nominal concentrations of 0.063 and 0.20 mg/L, determined in the experiment as the 21-day NOEC and LOEC. From the control samples, one sample per each sampling date was analyzed.
Vehicle:
no
Details on test solutions:
TEST WATER
The test was conducted in reconstituted water (Elendt M7 medium). Before use, the test water was aerated until oxygen saturation. During the test, the test media were not aerated.

MATERIAL
- The test was performed in 100-mL glass beakers containing 80 mL of test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

DOSAGE
Prior to the start of the test and prior to each test medium renewal, a stock solution of nominal 20 mg/L was freshly prepared by completely dissolving 40 mg of the test item (dosed in the range of 39.9 to 40.4 mg) in 2000 mL of test water using ultrasonification (5 minutes) and intense stirring (5 minutes). This intensively stirred stock solution was used in a series of dilutions to prepare the test media of all test item concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
The study was performed with females of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was
originally supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred at Harlan Laboratories in culture medium
identical to the medium used for the test . The temperature and light conditions were identical to those of the test.

The stock animals were maintained in one liter glass beakers filled with culture medium and were transferred twice a week to fresh medium. The
animals were fed normally three times a week with green algae of the species Scenedesmus subspicatus and with a fish food suspension.

The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.

The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first
brood progeny. At the start of the test, the test animals were less than 24 hours old.

The test method and test species, Daphnia magna, are recommended by the test guidelines.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
The water temperature was between 20 and 21 °C during the test period
pH:
the pH of the test media and control ranged from 7.5 to 8.0
Dissolved oxygen:
The dissolved oxygen concentrations were at least 7.5 mg/L
Salinity:
according to Elendt M7 medium (for details see table under "any other information on materials and methodes incl. tables"- below)
Nominal and measured concentrations:
- The following nominal concentrations were tested: 0.0063, 0.020, 0.063, 0.20 and 0.63 mg/L. Additionally, a control was tested in parallel (test water without test item).
- The measured test item concentrations in the freshly prepared test media of the nominal concentrations of 0.063 and 0.20 mg/L were between 62 and 95% of the nominal values at the start of the test medium renewal periods. In the stability control samples without food particles and daphnids, the measured concentrations were between 55% of nominal and below the limit of quantification (LOQ of the analytical method: 20.6 μg/L) at the end of the test medium renewal periods of 48 to 72 hours.
Details on test conditions:
EXPERIMENTAL CONDITIONS
- This semi-static test with test medium renewals every 48 or 72 hours was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water
temperature was maintained at 20-21 °C.
- A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was between approximately 520 and 680 Lux.
- In this semi-static test, the test media of all treatments were renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period (every Monday, Wednesday, and Friday). At these dates, the surviving test animals were carefully transferred by means of glass tubes from the old test vessels into the freshly prepared test medium.
- The test animals were fed daily (with the exception of Day 4, when daphnids were not fed) with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (freshly grown at Harlan Laboratories) and a fish food suspension.
- The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49304 Melle / Germany) in 500 mL
of test water. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test water and boiled. The suspension was stored deep frozen in small quantities until use.
- The carbon contents of the algal and fish food suspensions were determined using a Shimadzu TOC 5000A Analyzer. The food amount (based on the measured concentrations of the total organic carbon (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.

STUDY DESIGN
- The study was started with 10 daphnids per treatment. Each test animal was kept individually in a glass beaker. The test animals were randomly distributed to the test vessels. The test duration was 21 days.


Reference substance (positive control):
not required
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.022 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.13 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
BIOLOGICAL RESULTS:
- In the control and at the lower test concentrations up to and including 0.13 mg/L, the survival of the test animals at the end of the test was 100% . At the highest test concentration, all test animals were dead at observation on Day 12.
- The first young offspring released from their parent animals were recorded in the control and at all test concentrations up to and including 0.13 mg/L at observation on Day 9 . At the highest test concentration, no offspring were produced.
- The mean reproduction rate of the daphnids in the control was 101.1 ± 6.9 living offspring per adult (mean ± standard deviation). No significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the test concentration of 0.022 mg/L (Williams’ test, one-sided, alpha = 0.05). First at the concentration of 0.13 mg/L, the mean reproduction rate of surviving daphnids was statistically
significantly reduced to the mean of 52.9 ± 17.9 living offspring (52% compared to the control).
- With the exception of the reported mortality and the reduced reproduction rates, no visible abnormalities were observed at the test animals during
the test.
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test.
The mean reproduction rates of the daphnids at the test concentrations were compared to the control by multiple Williams’ tests .


Analytical results:

The measured test item concentrations in the freshly prepared test media of the nominal concentrations of 0.063 and 0.20 mg/L were between 62  and 95% of the nominal values at the start of the test medium renewal periods . In the stability control samples without food particles and daphnids,

the measured concentrations were between 55% of nominal and below the limit of quantification (LOQ of the analytical method: 20.6 μg/L) at the end of the test medium renewal periods of 48 to 72 hours.

In the stability control samples taken from the actual test including food particles, the measured concentrations at the end of the test medium

renewal periods were all <LOQ and thus lower than in the stability control samples without food particles. Thus, a part of the test item had obviously adsorbed onto the food particles. However, since filter feeding test animals as Daphnia may take up the test item also from ingested food, the

decrease of the test item concentrations in the test media due to adsorption onto food is not taken into account for calculation of the mean

measured concentrations.

The biological results were based on mean measured concentrations calculated as time-weighted means of the test item concentrations measured at the start of the test medium renewals and the concentrations measured in the stability control samples without food at the end of the renewal periods. The mean measured test concentrations were 0.022 mg/L (nominal 0.063 mg/L) and 0.13 mg/L (nominal 0.20 mg/L).

Water quality results:

During the test period, the pH of the test media and control ranged from 7.5 to 8.0 (Table 4). The dissolved oxygen concentrations were at least 7.5 mg/L. The water temperature was between 20 and 21 °C during the test period. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test medium renewal periods.

Validity criteria fulfilled:
yes
Conclusions:
Taking into account the effects on survival and reproduction of the test animals, the highest concentration of the substance tested without toxic effects after the exposure period of 21 days (21-day NOEC) was 0.022 mg/L. The lowest concentration tested with toxic effects (21-day LOEC) was determined to be 0.13 mg/L due to the statistically significantly reduced mean reproduction rate of Daphnia magna at this test concentration.
Executive summary:

The effect of the test item on the survival and reproduction of Daphnia magna was investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2008): “Daphnia magna Reproduction Test”, the EU Commission Directive 92/69/, C.20: “Daphnia magna Reproduction Test” and the Commission Regulation (EC) No 440/2008, C.20: “Daphnia magna Reproduction Test”.

 

The nominal concentrations tested were 0.0063, 0.020, 0.063, 0.20 and 0.63 mg/L. Additionally, a control was tested in parallel.

 

The concentrations of the test item were measured in the samples from the test concentrations of nominal 0.063 and 0.20 mg/L, determined in the experiment as the 21-day NOEC and LOEC. The measured test item concentrations in the freshly prepared test media of these test concentrations were between 62 and 95% of the nominal values at the start of the test medium renewal periods. At the end of the test medium renewal periods of 48 and 72 hours the measured concentrations were between 55% of nominal and below the limit of quantification (LOQ of the analytical method: 20.6μg/L).

The biological results were based on mean measured concentrations calculated as time-weighted means of the test item concentrations measured at the start and the end of test medium renewals. The mean measured test concentrations were 0.022 mg/L (nominal 0.063 mg/L) and 0.13 mg/L (nominal 0.20 mg/L).

 

Summary of the results after 21 days of exposure of the test animals:

 

 

Control

Substance
(nominal test item concentration in mg/L)

 

0.0063

0.020

0.063
(0.022)
#

0.20
(0.13)
#

0.63

Mortality (%) after 21 days of exposure

0

0

0

0

0

100

Mean reproduction rate (living offspring per surviving adult)

101.1

91.7

95.8

95.3

52.9 *

n.a.

Mean reproduction rate in % of control

100

91

95

94

52

n.a.

overall 21-day NOEC

0.022 mg/L (based on mean measured concentrations)

overall 21-day LOEC

0.13 mg/L (based on mean measured concentrations)

# : mean measured test item concentration

n.a.: not applicable (all parent animals died during the test)

* statistically significantly lower than the control value, results of a Williams’ test, one-sided,a= 0.05

Taking into account the effects on survival and reproduction of the test animals, the highest concentration of the substance tested without toxic effects after the exposure period of 21 days (21-day NOEC) was 0.022 mg/L. The lowest concentration tested with toxic effects (21-day LOEC) was determined to be 0.13 mg/L due to the statistically significantly reduced mean reproduction rate of Daphnia magna at this test concentration.

Description of key information

Taking into account the effects on survival and reproduction of the test animals, the highest concentration of the substance tested without toxic effects after the exposure period of 21 days (21-day NOEC) was 0.022 mg/L. The lowest concentration tested with toxic effects (21-day LOEC) was determined to be 0.13 mg/L due to the statistically significantly reduced mean reproduction rate of Daphnia magna at this test concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.022 mg/L

Additional information

An experimental study was available in order to assess the effect of the test item on the survival and reproduction of Daphnia magna. The study was investigated in a semi-static test over 21 days following the OECD Guidelines No. 211. The study was performed under GLP-conditions.

 

The biological results were based on mean measured concentrations calculated as time-weighted means of the test item concentrations measured at the start and the end of test medium renewals. The mean measured test concentrations were 0.022 mg/L (nominal 0.063 mg/L) and 0.13 mg/L (nominal 0.20 mg/L).

 

The summary of the results after 21 days of exposure of the test animals are reported in the following table:

 

 

Control

Substance
(nominal test item concentration in mg/L)

 

0.0063

0.020

0.063
(0.022)
#

0.20
(0.13)
#

0.63

Mortality (%) after 21 days of exposure

0

0

0

0

0

100

Mean reproduction rate (living offspring per surviving adult)

101.1

91.7

95.8

95.3

52.9 *

n.a.

Mean reproduction rate in % of control

100

91

95

94

52

n.a.

overall 21-day NOEC

0.022 mg/L (based on mean measured concentrations)

overall 21-day LOEC

0.13 mg/L (based on mean measured concentrations)

# : mean measured test item concentration

n.a.: not applicable (all parent animals died during the test)

* statistically significantly lower than the control value, results of a Williams’ test, one-sided,a= 0.05

The study satisfies guideline requirements for long-term toxicity to aquatic invertebrates studies and was therefore considered as acceptable for that endpoin

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