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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)
Version / remarks:
(1998)
Principles of method if other than guideline:
It was indicated that the study was conducted to fulfill the requirements of the Association Française de Normalisation AFNOR. No guideline was indicated but in principle, the test conducted is similar to the OECD TG 212 (adopted 1998), however a clearly defined concentration range allowing the determination of a NOEC was not indicated and not all parameters recommended by the TG were considered/reported (e.g. embryonic stage at test initiation, no data on appearance, behaviour and weight).
GLP compliance:
not specified
Analytical monitoring:
no
Details on sampling:
- Only qualitative analysis of ash, no quantitative analysis
- Sampling method: 15 samples were supplied by the sponsor to the Institut Pasteur de Lille for analyses. The samples were analyzed especially for metals (aluminium, chrome, nickel, selenium) and fluoride contents. Analyses were conducted on samples of Seine and synthetic water as controls, on the leachate of 100 g/L ashes (M’3) and on 1/4 and 1/16 dilution of the leachate.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For preparation of the different test solution, either synthetic water or water sampled from the Seine was used as solution medium. The samples M1 and M’1 (30 litres), M2 and M’2 (30 litres), N and N’ (60 litres) and the ashes (30 kg) were sampled on site and were supplied by the sponsor to the testing facility.They were stored at 12 to 14 °C in the dark.
The M3 and M’3 samples were prepared and obtained one day prior testing from a 87 g/L and a 100 g/L ash solution, respectively; the samples were stored as described above.
Old M1 and M2 samples refer to M1 and M2 samples stored for 47 days. The synthetic water was obtained from demineralized water supplemented with CaCl2-2H2O (284 mg/L), MgSO4-7H2O (123.3 mg/L), NaHCO3 (63 mg/L), KCl (5.5 mg/L); an oxygen content of ≥ 90% was assured. The test solutions were prepared daily and were monitored for pH, temperature and O2.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: carp
- Source: Installations Piscicoles Expérimentales (IPE) de l’INRA, Jouy-en-Josas, France
- Maintenance conditions: standardized and controlled (water quality, feed, environmental physical-chemical parameters)
- Temperature: the animals were maintained at a water temperature > 16 °C for 3 months (estival period) and received plenty of food. Thereafter they were transferred into water at a temperature < 12 °C until testing.
- Pre-exposure reproductive performance: no data

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): 8 days prior to ovulation, 3 males and 2 females were transferred from the IPE to the testing facility. The males and females were maintained separately in tanks (maximal charge: 1 kg bw /20 litres) at a temperature of 20 ± 0.5 °C in well-aerated water under flow-through conditions with a photoperiod of 14 hours. During the 4 days preceding ovulation, the animals were fasted to avoid contamination of the gametes by feces.
- Method of collection of fertilized eggs: forty hours prior ovulation, the animals of both sexes received a first injection of hypophysis extract (aqueous solution of 3 mg/mL of hypophysis extract in 7 g/L NaCl) at a dose level of 0.3 mg/kg bw; after 28 hours, they received a second injection, this time at a dose level of 7 and 3 mg/kg bw for the males and females, respectively. From the 10th hour following the second injection, ovocytes were collected from the females at 1 hour intervals until getting a sufficient number of gametes. Thereafter sperms were collected from the males. The gametes were obtained by abdominal massage; the animals were anaesthetized by bathing in 0.5 mL/L of 2-phenoxyethanol.
- Subsequent handling of eggs:
For fertilization, the gametes of one female were mixed with the semen of the 3 males. 10 µL of semen was added to the ovocytes and the mixture was then dispersed into each incubator containing 20 mL of test solution. After 3 minutes, the eggs adhered to the bottom of the incubator. After 10 minutes, the eggs were gently rinsed with 20 mL of test solution to eliminate excess of semen. Thereafter the eggs were incubated in 150 mL of the test solution for 7 days. The vessels containing the test solution were 250 mL polyethylene recipients with perforated caps. During incubation they were placed on an oscillating table. The test solutions were renewed and temperature, pH and dissolved O2 were monitored daily in each vessel.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
First test (M, N): 23.8 °C (mean of 7 days)
Second test (M’, N’): 24.5 °C (mean of 7 days)
pH:
First test (M, N): 7.5 (mean of 7 days)
Second test (M’, N’): 7.6 (mean of 7 days)
Dissolved oxygen:
First test (M, N): 6.38 mg/L (mean of 7 days)
Second test (M’, N’): 6.9 mg/L (mean of 7 days)
Nominal and measured concentrations:
Test concentrations (nominal):
M1: Effluents of ash retention basin of the central unit and dilution series
M2: Dilution water area for M1 effluents and dilution series
M3: Leachates of treated ash (87.5 g/L first series and 100 g/L second series) and dilution series
Details on test conditions:
TEST DESIGN:
First test series:
The first test series comprised 54 incubators designated as follows:
M1, M2 and M3 (undiluted)
M1/4-S, M2/4-S, M3/4-S (diluted to 1/4 in synthetic water (S))
M1/16-S, M2/16-S, M3/16-S (diluted to 1/16 in synthetic water (S))
M1/64-S, M2/64-S, M3/64-S (diluted to 1/64 in synthetic water (S))
15 synthetic water (S) controls were added
3 natural Seine water (N) controls were added.

Second test series:
The incubators in the second series were designated as follows:
M1, M2 old (stored for 47 days at 5 °C; undiluted)
M’1, M’2 (undiluted)
M’3-S and M’3-N (undiluted)
M’3/4-S and M’3/4-N (diluted to 1/4 in synthetic water (S) or normal water (N))
M’3/16-S and M’3/16-N (diluted to 1/16 in synthetic water (S) or normal water (N))
M’3/64-S and M’3/64-N (diluted to 1/64 in synthetic water (S) or normal water (N))

The test conduct was similar for both series tested.

TEST SYSTEM
- Test vessel: 250 mL polyethylene recipients with perforated caps
- Renewal rate of test solution (frequency/flow rate): daily
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 3

EFFECT PARAMETERS MEASURED
The total number of eggs used for incubation was designated as T (range: 50 to 150 fertilized eggs/replicate)
The number of surviving embryos after 1, 3, and 7 days of incubation was designated as V1, V3, and V7, respectively
The number of escaped embryos in the escape test at test ending (see below) was designated as HP
The measured parameters were as follows:
Fertilization rate and initial embryonal development
Survival rate at the end of hatching
Survival rate at the end of the embryonal development
Rate of larval mobility at the end of the embryonal development (escape test)
Growth, measured as the relative length (L) at the end of the the embryonal development
NOEC and LOEC

VEHICLE CONTROL PERFORMED: yes

VALIDITY CRITERIA
The validity of the results of the study is assured when following criteria are fulfilled:
- at least 60% survival of control embryos at day 7 (test ending)
- dissolved O2 in control vessels > 60%

STATISTCAL ASSESSMENT OF RESULTS: yes
Reference substance (positive control):
not specified
Details on results:
CONTROLS:
In controls, the survival of the larvae after 7 days was 89% in the first test series and 90% in the second one.

TESTS SAMPLES:
M1 and M’1 (effluents from the storage tank for ashes of the thermic power plant in Montereau, sampled on 10 February and 24 March 1993, respectively)
Neither survival nor mobility was affected by incubation with M1 and M’1 samples; an increased mortality was reported for one of the three M1/64 dilution replicates, as the two remaining replicates showed survival rates similar to control, the finding was considered to be incidental. A statistical significant decrease in larval growth and an alteration of the bottom adherence of the ovocytes were noticed during incubation with undiluted M1; no such effects were seen when dilutions of M1 and old sample (47 days of storage at 5 °C) were tested.

M2 and M’2 (water area used for dilution of the effluents M1 and M’1)
No effects were reported. In fact, a significant difference in length was observed between M2/64 and M2, which however was considered to be incidental as 2 replicates of 3 were lost accidentally and as in the third replicate, survival was 90%.

M3 and M’3 (leachates of treated ashes)
In group M3 (first test series, 87 g/L leachate, synthetic water) survival and mobility were inconspicuous but mean growth was reduced compared to control (6.2 cm versus 6.9 cm for control); in contrast, at the highest dilution level M3/64, mean growth was increased compared to control (7.2 cm versus 6.9 cm).

M’3-S (second test series, 100 g/L leachate, synthetic water) applied undiluted resulted in 100% mortality already at day 1 of incubation, thus survival was 0% versus 96% in control at day 1. It was assumed that the high mortality was related to the low pH of 4.19. In the remaining groups (M’3/4-S, M’3/16-S, M’3/64-S), survival at test ending was slightly below control level (respectively 80, 83 and 73% versus 90%); however, as no concentration-effect relationship was evident, the differences from control were considered to be of no relevance. In the M’3/4-S and the M’3/16-S group, growth was affected with a mean length of 6.1 and 6.5 cm versus 6.9 cm for control; in the M’3/64-S group, mean length was 6.6 cm. Mobility also was reduced in the 3 dilution groups, ranging between 43 and 53% versus 82% for control.

In the M’3-N (second test series, 100 g/L leachate, Seine water), survival at test ending was significantly reduced to 35% versus 89% in the control; growth was reduced, with a mean length of 5.8 cm versus 6.6 cm for control, and mobility was reduced to 45% (control: 81%). In the remaining groups (M’3/4-N, M’3/16-N, M’3/64-N), survival and growth were inconspicuous, i.e., within control range; mobility however was reduced in both, the M’3/4-N and the M’3/16-N groups (69% and 73%, respectively, versus 81% for control).

There were no effects on survival of fertilized eggs by effluents from the storage tank of a thermic plant unit and by diluted effluents from an inconspicuous water area. The only effects on egg survival could be observed with undiluted leachates (again no effects at 1/4 dilution of the leachates).

There were no effects on the mobility of newly hatched larvae (NOEC (7d) = effluent of storage tank or diluted effluent or 1/4 leachate of 100 g ash per 1 L water). The only effect on mobility of larvae was observed with leachates received with Seine water.

There were no effects on embryonal growth with 1/4 effluent from storage tanks, with 1/4 of the diluted effluent and with 1/4 of the leachate.

In particular survival rate, growth rate and mobility of the larvae were evaluated as parameters indicative of toxicity. The main findings can be summarized as follows:

M1 undiluted affected the bottom adherence of fertilized eggs and the larval growth; M’1 had no adverse effects on the early life stages of carp.

M2 had no adverse effects on the early life stages of carp.

M3 leachate from 87.5 g/L ashes obtained with synthetic water affected the growth of the larvae.

M3 leachate from 100 g/L ashes obtained with either synthetic or naturalSeinewater affected survival; even when diluted, effects were seen on growth and mobility.

The toxicity of the M3 leachates is suspected to be due to the monitored Aluminium, Nickel, Chrome and Sélenium contents.

No NOEC could be derived.
Validity criteria fulfilled:
yes
Conclusions:
A toxic potential of the leachates from ashes to Carp embryos/larvae could be demonstrated, but no NOEC could be derived.
Endpoint:
fish early-life stage toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
other information
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Remarks on result:
not determinable
Remarks:
No NOEC could be derived

Description of key information

Key value for chemical safety assessment

Additional information

According to column 2 of EC 1970/2006 Annex IX long-term toxicity testing shall only be proposed if the chemical safety assessment indicates the need to investigate further the effects on aquatic organisms.

Since no studies investigating the long-term toxicity to fish of cenospheres, fly ash (CAS 93924-19-7) are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to a structurally related ash (CAS 931-322-8) was conducted. This read-across is justified within the analogue justification in IUCLID Section 13.

For ashes (residues), coal one study was conducted to fulfill the requirements of the Association Française de Normalisation AFNOR. No guideline was indicated but in principle, the test conducted is similar to the OECD TG 212 (adopted 1998), however a clearly defined concentration range allowing the determination of a NOEC was not indicated.

In particular survival rate, growth rate and mobility of the larvae were evaluated as parameters indicative of toxicity.

Fertilized eggs from Cyprinus carpio were incubated for 7 days in test solutions at different dilution levels (undiluted, diluted to 1/4, 1/16 and 1/64) obtained from effluents from the storage tank for ashes of the thermic power plant in Montereau an inconspicuous water area used for dilution of effluents and leachates (87.5 g/L first series and 100 g/L second series) of treated ashes, either by Seine-water or by synthetic water.

There were no effects on survival of fertilized eggs by effluents from the storage tank of a thermic plant unit and by diluted effluents from an inconspicuous water area. The only slight effects on egg survival could be observed with undiluted leachates (again no effects at 1/4 dilution of the leachates). There were no effects on the mobility of newly hatched larvae (NOEC (7d) = effluent of storage tank or diluted effluent or 1/4 leachate of 100 g ash per 1 L water). There were also no effects on embryonal growth with 1/4 effluent from storage tanks, with 1/4 of the diluted effluent and with 1/4 of the leachate.

A definitive NOEC can not be derived because a clear concentration range is missing, however based on short-term tests from each trophic levels, the test substance can be regarded as acutely not harmful to aquatic organisms. Moreover, chronic studies for invertebrates and algae result in no classification; therefore and for animal welfare no further long term studies with fish are necessary.