Registration Dossier

Diss Factsheets

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 May - 16 June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations
- Sampling method: At the start of the exposure-renewal intervals (0 hours), samples of the fresh media were taken and analyzed after preparation of all concentrations and the control. At the end of the exposure-renewal intervals (48 or 72 hours), samples of the old media were taken from the test vessels.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock solution of 118 mg active acid/L was prepared with the culture medium without EDTA for each exposure interval (test item was weighed and added to dilution water, then agitated until the solution was clear). Test solutions were prepared by dilution of the stock solution with dilution water without EDTA.
- Controls:
dilution water without EDTA
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):
no evidence of undissolved material reported
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name:
water flea
- Strain/clone: Not reported
- Age at study initiation (mean and range, SD):
less than 24 hours old
- Weight at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD):
not reported
- Method of breeding:
daphnids were held in glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx.
- Source:
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of parental stock (mean and range, SD):
no first brood progeny was used for the test, no further details reported.
- Feeding during test
- Food type:
Pseudokirchneriella subcapitata (0.353 – 0.551 mL) and Desmodesmus subspicatus (0.346 – 0.627 mL) suspension
- Amount:
max. 0.2 mg C per Daphnia and day.
- Frequency: daily

ACCLIMATION
- Acclimation period:
at least 2 hours
- Acclimation conditions (same as test or not):
same as the test (dilution media without EDTA)
- Type and amount of food:
not reported
- Feeding frequency: not reported
- Health during acclimation (any mortality observed):
not reported
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
237 - 281 mg/L as CaCO3
Test temperature:
19.1 - 20.9 degrees C
pH:
6.41 - 8.08
Dissolved oxygen:
5.27 - 9.57 mg/l
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
Nominal: control, 3.93, 9.04, 20.8, 47.9 and 110 mg active acid/L
TWA mean measured: control, 3.91, 8.21, 19.1, 45.6, 106 mg active acid/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers (5 (ID) x 8 (H) cm)
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 100mL capacity, 50mL test volume
- Aeration: test vessels were not aerated during the test
- Renewal rate of test solution (frequency/flow rate): medium renewal of the test solutions was 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 culture medium
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Conductivity: not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water quality parameters (i.e. pH-values, dissolved oxygen concentration, total hardness and water temperature) were measured once within 7 days, in the fresh media at the start of an exposure-renewal interval (0 hours) and in the old media at the end of an exposure-renewal interval (48 or 72 hours), in one replicate of the control and the highest concentration of the test item with a living parent animal.
The water quality parameters in fresh media were measured in an additional replicate without daphnids of the highest concentration of the test item and the control. At the end of the exposure-renewal interval, the water quality parameters of the old media were measured in a test vessel of the highest concentration of the test item and the control, which contains daphnids and food algae.

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and immobilisation of daphnids were observed once a day at least. The first appearance of juveniles was checked daily, the number of neonates were counted once a day from the first appearance of neonates. The number of aborted eggs or dead offspring and the dates of observation were recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.3
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities:
behaviour of daphnids was observed each day of observation, but no abnormalities were reported
- Observations on body length and weight:
see Table 4
- Other biological observations:
no male daphnids or ephippia (winter eggs) were observed in the control or test groups during the test.
- Mortality of control:
0% mortality in the control at 7, 14 and 21 days
- Other adverse effects control: no adverse effects were observed in the control group
- Immobilisation of control: 0% immobilisation in the control group
- Abnormal responses:
some stillborn juveniles and aborted eggs were observed, see Table 3
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: not reported
Reported statistics and error estimates:
NOEC and LOEC values were determined using Dunnett's multiple t-test for reproduction and Fisher's exact Binomial test with Bonferroni correction for adult mortality. Data were analysed for normality and variance and homogeneity using Shapiro-Wilk's and Levene's tests, respectively.
ECx values were derived empirically from the observed data.

Table 1. LC-MS/MS analysis of the test item and equivalent active acid concentrations

Nominal concentration (mg test item/L)

Day 0 fresh medium 0 hours

(mg test item/L)

Day 2 old medium

48 hours

(mg test item/L)

Day 9 fresh medium

0 hours

(mg test item/L)

Day 12 old medium

72 hours

(mg test item/L)

Day 14 fresh medium

0 hours

(mg test item/L)

Day 16 old medium

48 hours

(mg test item/L)

TWA mean measured concentration (mg test item/L)

TWA mean measured concentration (mg active acid/L)

Control

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

Control

Control

10.0

9.92

11.0

8.20

11.0

10.2

9.65

9.94

3.91

23.0

21.4

22.6

17.7

21.2

20.2

24.2

20.9

8.21

52.9

51.4

43.1

44.6

49.6

52.1

54.2

48.7

19.1

122

117

95.2

114

121

135

113

116

45.6

280

288

260

263

258

311

238

269

106

Table 2. Mean reproduction and mortality ofDaphnia magnaadults after 21 days

TWA mean measured concentration (mg active acid/L)

Mean number of offspring per survived parental daphnid

Mortality

Mean

SD

CV

Day 7

Day 14

Day 21

Control

98.2

6.05

6.2

0

0

0

3.91

96.0

10.6

11.1

0

0

0

8.21

92.0

9.64

10.5

0

0

10

19.1

94.3

15.1

16.1

0

0

0

45.6

103

8.60

8.3

0

0

0

106

98.3

15.0

15.3

0

0

0

Table 3. Biological observations of reproduction inDaphnia magnaafter 21 days

TWA mean measured concentration (mg active acid/L)

Mean intrinsic rate of natural increase

Mean day of first appearance of living juveniles

Number of stillborn juveniles

Number of aborted eggs

Mean

SD

CV

Control

0.344

0.0252

7.3

8.3

44

0

3.91

0.343

0.0195

5.7

8.2

3

1

8.21

0.331

0.0190

5.7

8.7

3

0

19.1

0.331

0.0244

7.4

9.0

1

1

45.6

0.352

0.0193

5.5

8.0

1

0

106

0.333

0.0243

7.3

8.6

0

0

Table 4. Mean body length and weight of parent daphnids after 21 days

TWA mean measured concentration (mg active acid/L)

Mean length of survived parent daphnids (mm)

Mean dry weight of parent daphnids (mg)

Control

4.25

0.70

3.91

4.20

0.59

8.21

4.17

0.62

19.1

4.15

0.82

45.6

4.35

0.81

106

4.25

0.79

Validity criteria fulfilled:
yes
Remarks:
Mortality in the control group was 0% and the mean number of living offspring produced per introduced parent animal at the end of the test was 98 in the control group.
Conclusions:
21-day NOEC, EC10 and EC50 values of ≥106, >106 and >106 mg active acid/L have been determined for the effects of the test substance on the reproduction and mortality of Daphnia magna, based on time weighted average measured concentrations.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to Annex 3 of the CSR or IUCLID Section 13 for justification of read-across within the HEBMP category.
Reason / purpose for cross-reference:
read-across source
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 106 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: active acid
Basis for effect:
mortality

Description of key information

21-day NOEC and EC10 values of ≥106 mg active acid/L and >106 mg active acid/L have been determined for the effects of HEBMP-xNa on the reproduction and mortality of Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC10
Effect concentration:
> 106 mg/L
Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
>= 106 mg/L

Additional information

21-day NOEC, EC10 and EC50 values of ≥106, >106 and >106 mg active acid/L respectively have been determined for the effects of HEBMP-xNa on adult reproduction and mortality of Daphnia magna(Noack, 2021). The test concentrations were analysed by LC-MS/MS analysis and the results are expressed in terms of time weighted average (TWA) measured concentrations. The study was conducted according to OECD TG 211, in compliance with GLP and met the validity criteria, therefore it was assigned a reliability value of 1.

The acid and sodium salts in the HEBMP category are freely soluble in water. The HEBMP anion can be considered fully dissociated from its sodium cation when in dilute solution. Under any given conditions, the degree of ionisation of the HEBMP species is determined by the pH of the solution. At a specific pH, the degree of ionisation is the same regardless of whether the starting material was HEBMP-H, HEBMP-xNa, or another salt of HEBMP.

 

Therefore, when a salt of HEBMP is introduced into test media or the environment, the following is present (separately):

  1. HEBMP is present as HEBMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether HEBMP-xNa salt, HEBMP-H (acid form), or another salt was used for dosing.
  2. Disassociated sodium cations. The amount of sodium present depends on which salt was added.
  3. It should also be noted that divalent and trivalent cations would preferentially replace the potassium ions. These would include calcium (Ca2+), magnesium (Mg2+) and iron (Fe3+).