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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:
19.10.-20.11.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
see Any other information on materials and methods incl. tables
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: Fluidized Bed Combustion (FBC) Bottom Ash
- Substance type: inorganic
- Physical state: solid
- Main components: SiO2 - 28.69 %, Al2O3 - 15.87 %, Fe2O3 - 8.46 %, TiO2 - 5.04 %, CaO total - 27.58 %, MgO - 0.45 %, Na2O - 0.17 %, K2O - 0.36 %, MnO - 0.066 %, P2O5 - 0.40 %, SO3 - 9.64 %, CO2 - 1.00 %, Cl - 0.06 %, CaO free - 16.54 %, total As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn < 0.1%
- Lot/batch No.: FLP_ETI_2_6_2015
- Expiration date of the lot/batch: 05/2030
- Stability under test conditions: stable
- Appearance: Light grey powder with black particles
- Storage: The sample of the test substance has been stored in a dark box in original packaging

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
As the test substance is a multi-component poorly water-soluble substance, the water-accommodated fractions (WAFs) were prepared. Preparation of WAFs was carried out according to Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD Environmental Health and Safety Publication. Series on Testing and Assesment No. 23, Paris 2000. Section 3.11.)
The WAFs were prepared by adding measured amounts of the test substance to the M4 medium and then were stirred for suitable period. After period of stirring the WAFs were filtered through 0.45 μm filter and these solutions were used for testing.
The suitable period for achieving equilibrated concentration of dissolved and dispersed components in aqueous phase was determined in preliminary experiment. For preliminary experiment the WSFs were prepared separately as follows: the suspension of 100 mg of the test substance in 1 000 mL of M4 medium was stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, subsequently filtered through 0.45 μm filter. During this experiment the concentrations of metals (Al, Ca, Cu, Fe, K, Mg, Mn, Na, P, Si, Ti, Zn) in resulted WSFs and in the pure M4 medium were determined by atomic spectrometry method.
On the basis of the measured concentrations of metals, the period of 72 hours stirring was chosen as appropriate for the pretreatment of the test substance in the toxicity test. Non dissolved material was presented in the vessels after stirring. The non dissolved material present in WAFs has a potential to exert physical effects as encapsulation/entrapment of daphnids which are unrelated to toxicity. For this reason the all tested WAFs were filtered through 0.45 μm filter after stirring period to get water-soluble fractions for the exposure experiment.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna Straus were obtained by aperiodic parthenogenesis from own laboratory breeding. Daphnia were fed by a mixture of algae. The grow-up gravid females are separated into M4 medium. Young daphnia born in 24 hours are segregated by observation under a microscope. Only the females are transferred to the test containers.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none

Test conditions

Hardness:
2.50 - 2.80 mmol Ca+Mg /L
Test temperature:
19.5 - 21.0°C
pH:
7.6 - 8.4
Dissolved oxygen:
7.8 - 8.5
Salinity:
not applicable
Nominal and measured concentrations:
not applicable
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 ml
- Aeration: without
- Renewal rate of test solution (frequency/flow rate): 3× 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: 0.5 mg C/organism/day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Test medium: M4
- Conductivity: 1.46 – 2.53 µS/cm

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light
- Light intensity: 1250 – 1670 lx
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrations
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
The following data were recorded during the test: numbers of living offspring by each parent animal, presence of dead offspring and mortality parent animals. The total number of offspring per parent animal was calculated for each test vessel. If in any test vessel the parent animal died during the test, then the test vessel was excluded from the analysis.
The mean reproductive output for each loading rate and control were calculated. Reduction of reproductive output was expressed in %.
Results with reference substance (positive control):
a) Reference test
The sensitivity of the test species and correctness of test performance is periodically verified by testing with the reference substance, potassium dichromate. The results of the verification test with K2Cr2O7 are the following:
21 days – EC50 = 0.31 mg/L (95% confidence limit: 0.26 – 0.35 mg/L)

b) Data in the literature
HERMENS, JOOP, et al. Joint effects of a mixture of 14 chemicals on mortality and inhibition of reproduction of Daphnia magna. Aquatic Toxicology, Volume 5, Issue 4, November 1984, Pages 315-322.
21 days – EC50 = 0.27 mg/L

c) Method validity evaluation
The reduction in reproductive output of the test organism by the reference substance
(21d – EC50) obtained from our reference test is conformable to the values stated in the literature (Hermens et al., 1984).

Any other information on results incl. tables

Table No. 1: Number of offspring

10 mg/L

Vessel No.

(one parent animal/vessel)

Living offspring

Dead offspring

Dead parent animals

(day of death)

1

57

0

0

2

63

0

0

 3*

4

0

1 (14th day)

4

56

0

0

5

51

0

0

6

65

0

0

7

52

0

0

8

50

0

0

9

45

0

0

10

61

0

0

Mean number of living offspring

55.6

Total number of dead parent animals

1

1 mg/L

Vessel No.

(one parent animal/vessel)

Living offspring

Dead offspring

Dead parent animals

(day of death)

1

54

0

0

 2*

17

0

1 (12th day)

3

60

0

0

4

51

0

0

5

59

0

0

6

53

0

0

7

45

0

0

8

56

0

0

9

50

0

0

10

47

0

0

Mean number of living offspring

52.8

Total number of dead parent animals

1

0.1 mg/L

Vessel No.

(one parent animal/vessel)

Living offspring

Dead offspring

Dead parent animals

(day of death)

1

51

0

0

2

61

0

0

3

59

0

0

 4*

8

0

1 (14th day)

5

55

0

0

6

63

0

0

7

60

0

0

8

50

0

0

9

59

0

0

10

52

0

0

Mean number of living offspring

56.7

Total number of dead parent animals

1

control (M4)

Vessel No.

(one parent animal/vessel)

Living offspring

Dead offspring

Dead parent animals

(day of death)

1

63

0

0

2

60

0

0

3

68

0

0

4

57

0

0

 5*

1

0

1 (17th day)

6

59

0

0

7

62

0

0

8

65

0

0

9

63

0

0

10

54

0

0

Mean number of living offspring

61.2

Total number of dead parent animals

1

* - not included in calculation of means

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see Overall remarks
Conclusions:
The highest observed reduction in reproductive output of Daphnia was 13.7 % in the preliminary test. Therefore exact values of EL50 could not be calculated and the values of EL are given in the form of a range. The loading rates of the test substance were used for all evaluations and results.
Preliminary test showed that it could be reasonable to expect that estimated EL50 will be higher than 10 mg/L:
21 days–EL50 > 10 mg/L (loading rate)
In congruence with study plan, when the assumed value of EL50 is higher than 10 mg/L, no further testing is needed and the result obtained in preliminary test presents the result of whole study.
Executive summary:

The test substance, Fluidized Bed Combustion (FBC) Bottom Ash, was tested in reproduction test on Daphnia magna. The test was performed according to EU method C.20 – Daphnia magna Reproduction Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

Since the test substance is a multi-component poorly water-soluble substance the water-accommodated fractions (WAFs) were prepared by adding measured amounts of the test substance to the M4 medium and then were stirred for a period of 72 hours. After the period of stirring the WAFs were filtered through 0.45 μm filter and these water-soluble fractions (WSFs) were used for testing.

The preliminary test was performed in a range of the test substance loading rates 0.1 – 10 mg/L. The test was performed as semi-static, frequency of the test solutions renewal was three times a week. The highest calculated reduction in reproductive output was 13.7 %, the exact values of EL50 could not be calculated on the basis of data acquired. Test result is given in the form of a range. In congruence with study plan, when the assumed value of EL50 is higher than 10 mg/L, no further testing is needed and the result obtained in preliminary test presents the result of whole study.

The study was performed without the analytical determination of the test substance concentrations. Only the concentrations of metals in the loading rate 100 mg·L-1 were analysed in the preliminary experiment. Original purpose of metals determination was the choice of suitable period of stirring in the course of WSFs preparation.

For all evaluations and results the loading rates of the test substance were used.

Test results, based on nominal loading rate: 21 days–EL50 > 10 mg/L (loading rate).