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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Nov - 06 Dec 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Deficiencies in analytics and preparation of the test solutions: Analytical values for test item concentration only available for days 18-21. Broadly scattered measured concentration values due to difficulties in handling of the test item.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019
Reference Type:
study report
Title:
Unnamed
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz, Mainz, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Remarks:
HPLC-MS
Details on sampling:
- Concentrations: Control and 100 mg/L (Days 18 - 21)
- Sampling method: Samples were taken from freshly prepared and aged media

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Because of the very low solubility of the test item, water-accommodated fractions were prepared for the test. This was done by weighing the nominal load of 100 mg/L onto a cover glass, which was added to the corresponding amount of dilution water and shaken at 154 rpm for 3 days on an orbital shaker. The test item was applied in thin layers to achieve a large surface for contact with aqueous medium. The resulting unfiltered solution was used for the test. During validation it was found that filtration of the aqueous test item solutions must not be performed due to loss of test item at the filter material.
- Eluate: No
- Differential loading: No
- Controls: Dilution water without test item
- Evidence of undissolved material: The preparation of the test solution was very difficult. Despite the same handling, some solutions became cloudy and undissolved particles were visible. Other solutions remained completely clear. It was assumed that this is probably due to different surface characteristics of the test item applied to the cover glass. Differences in the surface result in different solution behaviour. It was also observed that the cover glass with the sticky test substance temporarily adhered to the glass wall of the flask and later came loose again. Due to the test item properties, it was very difficult to obtain reproducible measured values.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater flea (planktonic crustacean)
- Strain: Berlin
- Source: Umweltbundesamt in Berlin, Germany (27 Sep. 2007)
- Breeding conditions at test facility: The daphnids are bred year-round at the test facility in glass beakers with a nominal volume of 2 L using M4-medium (Elendt). The medium is renewed twice a week. Photoperiod: 16 h light/8 h dark, using neon tubes; Temperature: 20 ± 2 °C; Feeding: Green alga (Desmodesmus subspicatus)
- Age of daphnids at test start: Between 0 and 24 h
- Sex: Female
- Other: The animals used in the test were not first-brood of the respective parent animals (parthenogenesis). 22 h before the start of the test, the adult animals were separated from the young. Before the start of the test, the adults were caught with the help of a glass tube and the new-born daphnia, younger than 22 h, were sieved from the medium and immediately placed into a 250 mL beaker containing dilution water. After a settling-in period, animals with no apparent damage were used for the test.
- Feeding during test: Yes, daily
- Amount: 0.20 mg organic carbon/animal/day

ACCLIMATION
- Acclimation period: 22 hrs
- Acclimation conditions: Same as test
- Health during acclimation: Animals which showed no apparent damage were used for the test

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d

Test conditions

Hardness:
267 - 356 mg CaCO3/L
Test temperature:
19.6 – 23.4 °C
pH:
Blank control: 7.7 - 8.4
Treatment solution: 7.2 - 7.9
Dissolved oxygen:
Blank control: 8.2 - 9.9 mg/L
Treatment solution: 7.4 - 9.4 mg/L
Nominal and measured concentrations:
Control and 100 mg/L (nominal)
0.26 µg/L (Day 18, slightly below the LOQ but clearly identifiable), 14.1 mg/L (Day 19), 0.73 µg/L (Day 20) (limit concentration measured in new media)
not detectable (Day 19), < LOQ (Day 20) and not detectable (Day 21) (limit concentration measured in old media)
Broadly scattered values due to difficulties in handling of the test item.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (nominal volume 100 mL)
- Aeration: None
- Renewal of test solution: Daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Fill volume: 80 mL test solution/animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4-medium, according to guideline
- pH range: 7.7 - 8.4
- O2 concentration: ≥ 8.2 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 1000 lux

EFFECT PARAMETERS MEASURED:
- Number of offspring was counted (daily)

TEST CONCENTRATIONS
- Range finding study: No
Reference substance (positive control):
not specified

Results and discussion

Effect concentrations
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Water Accommodated Fraction
Basis for effect:
reproduction
Details on results:
- Behavioural abnormalities: No
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: Due to the test item properties it was very difficult to obtaine reproducible measured values. Despite the same handling, some solutions became cloudy and undissolved particles were visible. Other solutions remained completely clear. In the clear solutions, only very low concentrations were detectable in the freshly prepared solutions. In the turbid solutions, the concentration was many times higher (by several orders of magnitude), because undissolved substance was also measured.
- Effect concentrations exceeding solubility of substance in test medium: Yes
Reported statistics and error estimates:
No toxicity was observed. The number of offspring in the treatment was higher than in the control. Therefore, no statistical evaluation had to be performed.

Any other information on results incl. tables

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the guideline (Table 2).

Table 1: Validity criteria for OECD 211. 

Criterion from the guideline

Outcome

Validity criterion fulfilled

The mortality of the parent animals in the control (femaleDaphnia) does not exceed 20% at the end of the test.

Mortality of parent animals in the control was 0%.

Yes

The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60 in the control.

Mean surviving offspring in the control was 105 in the control.

Yes

 

ANALYTICAL DATA

Due to insufficient stability of the test item, medium renewal was performed every day. Therefore, a total of 21 fresh solutions were prepared. Despite the same handling, some solutions became cloudy and contained undissolved particles while other remained completely clear. It was assumed that this is probably due to differences to surface characteristics of the test item applied to the cover glass. Differences in the surface result in different dissolution behaviour. It was also observed that the cover glass with the sticky test substance temporarily adhered to the glass wall of the flask and later came loose again.

Validation showed that the solubility of the test item in daphnia medium is very low. Test item/water mixtures, treated with high-speed shaking, resulted in turbid mixtures. Test item/daphnia medium mixtures with a nominal load of 100 mg/L were shaken with low speed (100 rpm). The supernatants of the mixtures were sampled several times but did not result in measurable solutions. The validation shaken with slightly higher speed (150 rpm) for 3 d resulted in measurable concentrations of 1.27 µg/L. It was also found that filtration of the aqueous test item solutions must not be performed due to losses of the test item to the filter material. Therefore, for the preparation of test solutions in this study, the test item was added onto a cover glass and then added to the test medium and slowly stirred at 154 rpm for 3 d until a measurable concentration was reached.

The actual test item concentrations in the test solutions were determined by LC-MS. Originally, the test item concentrations were to be measured at least once a week in the same batch of the freshly prepared solutions (0 h) and the solutions on the next day (24 h). However, due to a device failure, the samples were only measured on Day 18 to 21 using a different LC-MS-MS device. The measured concentrations broadly scattered due to the difficulties encountered in the preparation of test solutions.

Due to the test item properties described above, it was difficult to obtain reproducible measured values. In the clear solutions, only very low concentrations were detectable in the freshly prepared solutions. In the turbid solution, the concentration was many times higher (several orders of magnitude) because undissolved substance was also measured. For medium renewal, this solution was used after a settling period of 2 h without undissolved particle. The measured concentrations showed the presence of dissolved test item in the test solution partly above the limit of solubility (Table 2).

Table 2. Measured test item concentrations in new (0 h) and old (24 h) test media on Days 18 – 21.

Nominal concentration

measured concentration

Day 18

Day 19

Day 20

Day 21

[mg/L]

new

new

old

new

old

old

Blank control

--

--

--

--

--

n.d.

100

0.26 µg/L*

14.1 mg/L

n.d.

0.73 µg/L

< LOQ

n.d.

n.d. = not detectable

* slightly below the LOQ but clearly identifiable

LOQ = limit of quantification (0.27 µg/L)

According to the OECD guidance document 23, results can be expressed in terms of nominal concentrations for test with chemicals that cannot be quantified by the most sensitive analytical methods at relevant concentrations (chapter 9, p.59). Therefore, the results in this study were based on the nominal concentrations.

 

BIOLOGICAL DATA

No toxicity was observed. The number of offspring the treatment was higher than in the control. Therefore, no statistical evaluation was performed. The effect values are summarized in Table 3.

Table 3. Summary of effect values after 21 d.

Parameter

Value

NOEC

≥ 100 mg/L

LOEC

> 100 mg/L

EC50

> 100 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.