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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:
21 Oct - 11 Nov 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.096, 0.24, 0.60, 1.50 and 3.75 mg TI/L (nominal); 9.8, 24.5, 61.1, 153 and 382 µg B/L (nominal)
- Sampling method: At test start and at each renewal samples of all treatments were taken from the test solution preparation just before distributing it to the 10 replicates. At the renewals samples of the aged test solution were taken from each vessel, pooled per concentration and measured.
- Sample storage conditions before analysis: Since analysis could not be applied immediately after sampling samples were stored in a refrigerator stabilized with HNO3 until analysis.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions were prepared by applying the Water Accommodated Fraction (WAF) approach according to the OECD guidance No. 23. To achieve the maximum possible test concentrations in the eluate, it was prepared (loading of 3.75 mg/L and 1.5 mg/L) in a polycarbonate vessel and was stirred for about 24 h at room temperature. Subsequently, the test preparation was filtered by using a 0.2 µm filter (PES (polyethersulfone)) bottle top filter, separate insoluble parts from the aqueous phase. The loadings of 0.096, 0.24, and 0.60 mg test item per litre were prepared by applying a dilution series starting with the filtrated clear test solution of loading 1.5 mg/L. The test solution was freshly prepared before test start and renewal.
- Controls: Dilution water control
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Waterflea
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene; Specimens used in the test are bred in the laboratory of the Fraunhofer IME.
- Age of parental stock (mean and range, SD): 4 - 24h
- Feeding during test
- Food type: Desmodesmus subspicatus and ArtemioFluid in a ratio of 9 : 1 (vol/vol)
- Amount: 0.2 mg C/Daphnia/day.

Breeding
- Breeding conditions (same as test or not): Adult Daphnia, at least 3 weeks old, are separated from the stock population by sieving. Batches of 30 to 50 animals are held at room temperature in ca. 1.8 L dilution water for one week. During this week the daphnids are fed daily with an algal suspension (Desmodesmus subspicatus) and LiquizellR (HOBBY). Algae growing in the log-phase are centrifuged and the pellet is re-suspended in a few mL of medium. 30 mL of this suspension is given to 1 L Daphnia medium. The water is changed once per week. Newborn Daphnia are separated by sieving, the first generation is discarded.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.4 - 2.9 mmol/L
Test temperature:
20.4 - 20.7°C
pH:
7.5 - 8.7
Dissolved oxygen:
7.4 - 10.7 mg/L
Nominal and measured concentrations:
nominal: 0.096, 0.24, 0.60, 1.50, 3.75 mg TI/L; 9.8, 24.5, 61.1, 153 and 382 µg B/L
measured (TWM): 9.91, 29.7, 84.8, 214, 494 µg TI/L; 1.01, 3.02, 8.64, 21.9 and 50.3 µg B/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Polypropylene beakers (120 mL nominal volume)
- Type (delete if not applicable): Covered with glass panes
- Material, size, headspace, fill volume: 50 mL test solution
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): three times weekly
- 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: ISO test water according the OECD guideline 202
- Intervals of water quality measurement: Oxygen concentration, pH value, and temperature are checked directly before adding the animals and at each water renewal in new and aged test solutions. Hardness is checked once a week at water renewal in new and aged control media and test solutions of the highest treatment.

OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: 943 – 990 lx (corresponding to 12.6 – 13.2 µE/(m² *s))

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The numbers of immobile daphnids were visually determined daily and the immobile daphnids were removed. Any abnormalities in appearance and behaviour were recorded if occurred.
The newborn daphnids per beaker were counted and removed daily until all daphnids start to reproduce (at least 10 days and at a maximum of 14 days). After that newborn daphnids were counted and removed three times weekly at each water renewal. Any abnormalities in condition (including male sex) or presence of winter eggs were checked and recorded.
At study termination, length of the adults up to the highest treatment without significant mortality was measured by digital photography and image analysis.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 3.75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 3.75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 3.75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: intrinsic
Details on results:
- Mortality of parent animals: no
- Other biological observations: No other clinical signs were observed in any replicate at any concentration tested. Neither any physical nor pathological symptoms were obtained.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24h) = 1.32 mg/L (August 2015)
Reported statistics and error estimates:
Statistical evaluations were done with version 3.2.1 of ToxRat® Professional by ToxRat® Solutions GmbH.

Adult body length exhibited no significant differences between treatments.

Age at the first brood was between 8.5 and 9.0 days up to the highest concentration tested. The difference was not statistically significant.

The cumulative number of offspring per replicate ranged from 71.5 to 88.2 in the control and all treatment assays. The difference of the number of offspring was not statistically significant. The intrinsic rate of increase ranged from 0.321 to 0.337 (mean values) in the control and all treatment assays. The difference was not statistically significant.

With 943 – 990 lx (corresponding to 12.6 – 13.2 µE/(m² *s)) the light intensity was below the maximum value of about 1125 - 1500 lx (15–20 µE/(m² *s)) recommended by OECD guideline 211. As light intensity was similar in all treatments, results are comparable.

Concentrations of the boron orthophosphate [Time weighted mean measured boron concentrations [µg B/L], time weighted mean recalculated test item concentrations [µg TI/L], and percentage of nominal concentration [%], TI = test item]

Nominal test item loading

0.096 mg TI/L

0.24 mg TI/L

0.60 mg TI/L

1.50 mg TI/L

3.75 mg TI/L

Nominal B loading


9.8 µg B/L


24.5 µg B/L


61.1 µg B/L


153 µg B/L


382 µg B/L

Time weighted mean measured B conc.

1.01 µg B/L

3.02 µg B/L

8.64 µg B/L

21.9 µg B/L

50.3 µg B/L

Time weighted mean recalculated test item concentration

9.91 µg TI/L

29.7 µg TI/L

84.8 µg TI/L

214 µg TI/L

494 µg TI/L

% of nominal

10.3

12.4

14.1

14.3

13.2

 

Survival, growth and reproduction data[SD = standard deviation. Number of D. magna per concentration: n = 10. TI = test item. TWM = Time weighted mean.]

Concentration

Parental survival

Growth
(length on day 21)

Age at first brood

Cumulative offspring per female

Intrinsic rate of increase

(Nominal)

(TWM)

(%)

Mean±SD
(cm)

Mean±SD
(days)

Mean±SD
(Ind.)

Mean±SD
(Ind./day)

Control

Control

100

0.51±0.05

8.9±0.5

   88.2±18.8

0.337±0.028

9.8 µg B/L
0.096 mg TI/L

1.01 µg B/L
9.91 µg TI/L

100

0.51±0.06

9.0±0.5

   84.2±  9.3

0.321±0.026

24.5 µg B/L
0.24 mg TI/L

3.02 µg B/L
29.7 µg TI/L

100

0.50±0.05

8.9±0.5

   79.3±32.5

0.327±0.034

61.1 µg B/L
0.60 mg TI/L

8.64 µg B/L
84.8 µg TI/L

100

0.54±004

9.0±0.5

   83.5±15.1

0.329±0.029

153 µg B/L
1.50 mg TI/L

21.9 µg B/L
214 µg TI/L

100

0.47±0.07

8.8±0.7

   71.5±12.1

0.322±0.021

382 µg B/L
3.75 mg TI/L

50.3 µg B/L
494 µg TI/L

100

0.50±0.06

8.5±0.0

  86.3±10.9

0.336±0.014

 

Effect summary table[Based on concentrations calculated from time weighted mean concentrations (TWM). n.d. = not determined due to mathematical reasons. TI = test item.]

Concentration

Parental survival

Growth
(length on day 21)

Age at first brood

Cumulative offspring per female

Intrinsic rate of increase

NOEC

≥ 50.3 µg B/L
[≥ 494 µg TI/L]

≥ 50.3 µg B/L
[≥ 494 µg TI/L]

≥ 50.3 µg B/L
[≥ 494 µg TI/L]

≥ 50.3 µg B/L
[≥ 494 µg TI/L]

≥ 50.3 µg B/L
[≥ 494 µg TI/L]

 

 

 

Validity criteria fulfilled:
yes

Description of key information

Key value for chemical safety assessment

Additional information

The influence of Boron orthophosphate (CAS 13308-51-5) on long-term toxicity of aquatic invertebrates of the species Daphnia magna was investigated in a semi static OECD 211 test under GLP conditions.

The highest test solution with 3.75 mg/L nominal test substance concentration was freshly prepared by addition method and serial dilution were prepared with supernatant of 1.5 mg/L of nominal test substance concentration in order to obtain the following desired test concentrations: 0.096, 0.24, 0.60, 1.50 mg/L. Daphnia magna was exposed to test solutions for 21 days. The boron concentrations in the test solutions were determined analytically. The mean measured boron concentrations were 1.01, 3.02, 8.64, 21.9 and 50.3 µg B/L. During the exposure period of 21 days no effects on survival, growth and reproduction of Daphnia magna were recorded. The NOEC is ≥ 3.75 mg /L (nominal test substance concentration) and ≥ 494 µg boron/L (measured concentration).