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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:
3 Okt 2017 - 27 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Bern, Switzerland
Analytical monitoring:
yes
Remarks:
HPLC/UV
Details on sampling:
- Concentrations: control, 3.6 and 10 mg/L (one of the duplicate test medium samples was analyzed)
- Sampling method: duplicate samples from the test media of all test concentrations and from the control were taken at the start of the test (Day 0) and at seven further sampling dates distributed over the test period.
- Sample storage conditions before analysis: All 10-mL samples were acidified with 200 µL hydrochloride acid to approximately pH 4 and were stored deep-frozen (at about –20 °C).The test item was stable under these storage conditions in a pre-test. For verification of the test item concentrations during the test period, a part of the samples was analyzed before test termination.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The application solutions were freshly prepared three days before start of the test (the dosing system was started at Day -3 before test start for equilibration) and thereafter four times during the test period (Days 3, 7, 13 and 20). For the preparation of the application solution used for the dosage of the highest test concentration, adequate volumes of the test item were pipetted into purified water at a nominal concentration of 1000 mg/L. The test item was completely dissolved by stirring for one hour. As the test item was volatile, stirring was performed in closed stirring vessels which were nearly completely filled with a volume of approximately 2.3 L purified water and which were closed by a glass. This highest application solution was diluted to prepare the respective lower test item concentrations. The pH of the application solutions was adjusted to pH 4 with hydrochloride acid for stabilisation. A pre-experiment showed that the solutions were stable during a period of at least 7 d under these conditions. The application solutions were continuously stirred and protected from light during their use of in maximum 7 d.
- Differential loading: yes
- Controls: Test medium without test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus
- Source: Laboratory culture (originally from the Daphnia Collection of the University of Basel/Switzerland).
- Age of parental stock (mean and range, SD): Daphnids used for the test originated from parental daphnids that were at least 14 d 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 h old, by removing the offspring <24 h before start of the test.
- Feeding during test: yes
- Food type: suspension of green algae of the species Desmodesmus subspicatus
- Amount and Frequency: 0.3 mg C per day and Daphnia, extended to 0.4 mg C per day at Day 7 as the parental daphnids had increase in size. Immediately prior to addition of the test medium aliquot to the test vessel, an aliquot of nominal 100 µL algae suspension was added to the funnel of the test vessel and was then washed into the test vessel by the 5.2 mL-aliquot of test medium (for details see field "details on test conditions").

ACCLIMATION
- Acclimation period: not necessary since cultivation conditions (medium, temperature and light) same as test conditions.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
20 °C
pH:
7.5 - 7.8
Dissolved oxygen:
9 - 10.8 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 0.16, 0.46, 1.3, 3.6, and 10 mg/L
Arithm. mean measured: n.a, n.a., n.a., n.a., 3.2, 9.3 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: flow-through glass beakers (outlet of the vessels was fitted with a stainless steel net to retain the parental daphnids and the offspring in the test vessel).
- Type: closed
- Fill volume: 50 mL
- Aeration: no
- Type of flow-through: The test vessel was covered with a funnel, which directed the new test medium to the bottom of the test vessel. The new test medium was directed to the bottom of the test vessel displacing the old test medium to the overflow at the top of the test vessel. The flow-through test vessels were replaced at least every three days or at the date when the adults were separated from the offspring.
- Renewal rate of test solution (frequency/flow rate): 0.75 mL application solution was added by use of a syringe pump into a full glass mixing chamber filled with 75 mL test water. The test medium was then stirred for approx. 3 min. The test medium was then directed to the 10 replicates by a Teflon tube, which was moved by a computer controlled extension arm from one replicate to the next. To each replicate test vessel an aliquot of 5.2 mL test medium was added. Thus, the test medium flow was the same for all replicate test vessels. The frequency of test medium preparation cycles was 8 cycles per hour. Given a test medium aliquot of 5.2 mL (distributed to each replicate test vessel), a number of 192 test medium preparation cycles per day and a test vessel volume of 50 mL, the test medium flow applied resulted in an approximately 20-fold test medium exchange rate per day. For the control, purified water adjusted to pH 4 was dosed in the same way as described for the test item treatments to simulate identical experimental conditions for the control.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guideline (Elendt M7 medium)
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water temperature, oxygen concentration and pH were measured alternately in one of the replicates of all test concentrations with surviving daphnids and in the control at the start of the test and two times per week thereafter. Additionally, the water temperature was monitored continuously by a data logger. The appearance of the application solutions and the test media in the mixing vessels and test vessels was checked each working day.


OTHER TEST CONDITIONS
- Photoperiod: 16-h light / 8-h dark cycle (30 min ransition period)
- Light intensity: 8 - 9 µE m^-2 s^-1

EFFECT PARAMETERS MEASURED: Immobility of adults or other visible abnormalities were observed daily. On the same dates, the test replicates were observed for the number of living and dead offspring and for the presence of aborted eggs. Offspring was separated from the adult at the day of observation by transferring the adult daphnia in a new test vessel by means of a wide bore glass pipette. The offspring was counted and was disposed thereafter. The body length of surviving adults was determined at the end of the test by measuring the daphnids from the top of the head to the basis of the spina with the use of a binocular microscope. The adults were disposed after evaluation.

TEST CONCENTRATION
- Range finding study: yes (no further details reported)
Reference substance (positive control):
not specified
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival/growth/reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
9.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: No visible abnormalities were observed at the surviving test animals during the test period.
- Observations on body length and weight: The mean body length of the daphnids in the control was 4.78 ± 0.11 mm. No inhibitory effect of the test item on the body length was determined up to and including the test concentration of 3.2 mg/L. At the highest test concentration of 9.3 mg/L, no body length could be determined since no parental daphnids had survived until the end of the test.
- Mortality of control: 10% mortality in the control
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
A statistical evaluation of the data for the mean reproduction rates and the mean body lengths of the daphnids was not performed due to the clear results, i.e. the corresponding values at nominal 3.6 mg/L were slightly higher compared to the control.

ANALYTICAL RESULTS:

The analytical results showed, that under the flow-through conditions the concentrations of the instable test item could be maintained sufficiently constant during the test period of 21 days.

Table 1: Arithm. Mean Measured Concentrations of Test Item.

Nominal Concentration of Test Item [mg/L] Arithm. Mean Measured Concentrations of Test Item [mg/L] % of Nominal Concentrations
0.16 n.a. n.a.
0.46 n.a. n.a.
1.3 n.a. n.a.
3.6 3.2 89
10 9.3 93

BIOLOGICAL RESULTS:

Table 2: Number of Surviving Daphnids over the test period.

Exposure Time [Days] Number of Surviving Test Animals
Nominal Test Item Concentration (Mean Measured Test Item Concentration) [mg/L]
Control 0.16 0.46 1.3 3.6 10
(n.a.) (n.a.) (n.a.) (n.a.) -3.2 -9.3
0 10 10 10 10 10 10
4 10 10 10 10 10 10
5 10 10 10 10 10 9
6 10 10 10 10 10 9
7 10 10 10 10 10 6
9 10 10 10 10 10 6
10 10 10 10 10 9 3
11 9 10 10 10 9 3
12 9 10 10 10 9 2
14 9 10 10 10 9 2
15 9 10 10 10 9 1
16 9 9 10 10 9 1
17 9 9 10 10 9 0
21 9 9 10 10 9 0
% Surviving on Day 21 90 90 100 100 90 0

Table 3: Number of Offsprings

Exposure Time [Days] Number of Living Offspring 
Nominal Test Item Concentration (Mean Measured Test Item Concentration) [mg/L]
Control 0.16 0.46 1.3 3.6 10
(n.a.) (n.a.) (n.a.) -3.2 -9.3
1 208 191 184 162 232 *
2 161 206 208 177 183 *
3 184 212 185 187 * *
4 161 187 213 175 214 *
5 * 169 212 180 152 *
6 178 180 197 185 173 *
7 189 182 158 163 196 *
8 184 185 223 227 180 *
9 223 214 177 222 202 *
10 173 * 175 196 214 *
Mean 186 192 193 187 194
± SD 23 16 21 22 25
n 10 10 0
CV % 12.3 8.1 10.7 11.8 12.7
% of Control 103 104 101 104
STAT # # # #

SD: Standard Deviation

CV %: Coefficient of variation: (SD/mean) × 100 %

*: Daphnia died during the test

°: The mortality of 10% was considered not to be due to a toxic effect of the test item. The calculation of the mean reproduction rate is therefore based on the surviving adults (according to the OECD Guideline No. 211, 2012).

#: A statistical evaluation of inhibition of the reproduction rate was not performed as the values obtained for the test item treatments were slightly higher compared to the control.

Further effect values:

LOEC (21 d) = 9.3 mg/L (mean measured)

EC10/50 (21 d) > 3.2 mg/L (reproduction rate and body lenth)

VALIDITY CRITERIA:

Table 4: Validity criteria for OECD 211.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The mortality of the parent animals (female Daphnia) does not exceed 20% at the end of the test.

10 % mortality in control

yes

The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60.

mean number of living offspring in the control was 186 per surviving adult

yes

The coefficient of variation around the mean number of living offspring produced per parent animal in the control should be =< 25 %

coefficient of variation was 12.3 % in the control

yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
The study, showed that the substance inhibits the growth and reproduction of the tested organism Daphnia magna after 21 d of exposure (OECD 211). A NOEC (21 d) value of 3.2 mg/L (arithm. mean meas.) was determined.

Executive summary:

The impact of the test item on the survival, growth (body length), and reproduction rate of aquatic invertebrates was investigated in a flow-through test over 21 days following the OECD Guideline 211 and GLP. The test organism Daphnia magna was exposed to nominal test item concentrations of 0.16, 0.46, 1.3, 3.6, and 10 mg/L and a control (10 organisms per concentration and control). A 20-fold test medium exchange rate per day was applied. The concentrations of nominal 3.6 and 10 mg/L were analytical monitored by HPLC/UV. The analytical results showed that the test item could be maintained sufficiently constant during the test period of 21 days, since 77 – 98% of the nominal test item concentrations were analytically recovered. The biological results were expressed in terms of arithmetic mean measured concentrations. Taking into account the effects of the test item on survival, growth, and reproduction of the Daphnids, the overall 21 day NOEC was 3.2 mg/L (arithm. mean measured). Additionally with respect to mortality a LOEC of 9.3 mg/L (arithm. mean) was determined.

Description of key information

NOEC (21 d) = 3.2 mg/L (arithm. mean meas., D. magna, OECD 211)

Key value for chemical safety assessment

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

Additional information

The impact of the test item on the survival, growth (body length), and reproduction rate of aquatic invertebrates was investigated in a flow-through test over 21 days following the OECD Guideline 211 and GLP. The test organism Daphnia magna was exposed to nominal test item concentrations of 0.16, 0.46, 1.3, 3.6, and 10 mg/L and a control (10 organisms per concentration and control). A 20-fold test medium exchange rate per day was applied. The concentrations of nominal 3.6 and 10 mg/L were analytical monitored by HPLC/UV. The analytical results showed that the test item could be maintained sufficiently constant during the test period of 21 days, since 77 – 98% of the nominal test item concentrations were analytically recovered. The biological results were expressed in terms of arithmetic mean measured concentrations. Taking into account the effects of the test item on survival, growth, and reproduction of the Daphnids, the overall 21 day NOEC was 3.2 mg/L (arithm. mean measured). Additionally with respect to mortality a LOEC of 9.3 mg/L (arithm. mean) was determined.