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Ecotoxicological information

Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Method: other: Early Life Stage Test based on a Procedure of the Committee on Methods for Toxicity Tests with Aquatic Organisms
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
MONITORING OF TEST SUBSTANCE CONCENTRATION: 
- weekly, 50 ml per concentration 
Vehicle:
no
Details on test solutions:
STOCK AND TEST SOLUTION AND THEIR PREPARATION
- Vehicle, solvent: not used
- Other procedures: diluter cycled for >= 24 h in advance
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISMS
- Supplier: inhouse (Bionomics Marine Research Laboratory)
- Wild caught: mothers from Big Lagoon (estuary of Gulf of Mexico)
- Post-hatch transfer time: eggs within 4 h after visual confirmation of  fertilization; embryos daily
- Pretreatment: egg production of mother females enhanced by injections  of human chorionic gonadotropin on 2 consecutive days; fertilization by  
addition of sperm suspension made from macerated testes excised from adult male fish
- Feeding during test: live Artemia salina nauplii daily
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
28 d
Test temperature:
- Test temperature: 29 +/- 1 °C
pH:
- pH: 7.8-8.3
Dissolved oxygen:
- Dissolved oxygen: 4.0-6.8 mg/l
Salinity:
- 22-28 per mil
Nominal and measured concentrations:
nominal: 0 / 30 / 59 / 119 / 238 / 476
measured: n.d. / 18 / 40 /  80 / 156 / 287
Details on test conditions:
TEST SYSTEM
- Test type: flow-through
- Concentrations: (mg/l)
- Dosing rate: 1 l per cycle
- Renewal of test solution: 4-7 cycles per hour
- Exposure vessel type: glass aquariums 30 cm wide, 30 cm high, 91 cm  long, containing approximately 41 l solution; 
- embryos in 100 ml glass  jars with 425 um-square mesh nylon screens as bottoms, suspended in  aquariums;
-  juveniles in glass chambers 14 cm x 20.5 cm x 26 cm with 425  um-square mesh nylon screen over one end 
- Number of replicates, fish per replicate: 2 replicates with 50 eggs each; 2 replicates with maximum 40 juveniles each
- Adjustment of pH: no
- Intensity of irradiation: approximately 1100 lux incident to water  surface
- Photoperiod: 16 hours light, 8 hours dark
- Controls: dilution water

DILUTION WATER
- Source: natural sea water, Big Lagoon, filtered
- Salinity: daily (few exceptions)
- pH: daily (few exceptions)
- Oxygen content: daily (few exceptions)

ENDPOINTS ASSESSED: 
- hatching success, survival, growth

STATISTICAL ANALYSIS: 
- statistical significance determined by analysis of  variance (ANOVA), after arcsin percentage transformations of binomial  percentages to angles of 
equal information in degrees (hatching success,  mortality); ANOVA (length); 95 % confidence level (p < 0.05) applied to differences between test and control solutions.
Reference substance (positive control):
not specified
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
80 mg/L
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
156 mg/L
Details on results:
STABILITY OF THE TEST CHEMICAL SOLUTIONS: 
- analytical concentrations  averaged only 64 % of nominal concentrations, possibly due to losses by  volatilization

mortality   hatching    length
control       4 %        83 %      11 mm
18 mg/l       0 %        86 %      11 mm
40 mg/l      18 %        83 %      12 mm
80 mg/l       8 %        79 %      11 mm
156 mg/l     25 %        75 %       4 mm
287 mg/l    100 %        20 %       -
NOEC / LOEC (hatching) = 156 mg/l / 287 mg/l
- Increased length at 40 mg/l may be due to increased availability of  food caused by high mortality.
- Adverse effects: 
  one two headed embryo at 18 mg/l, died after 1 day
  one eyeless juvenile at 40 mg/l, survived

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Method: other: Flow-Through Early Life Stage Test, US EPA proposal
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
MONITORING OF TEST SUBSTANCE CONCENTRATION: 
- 1st day, twice weekly, last  day
Vehicle:
no
Details on test solutions:
STOCK AND TEST SOLUTION AND THEIR PREPARATION
- Vehicle, solvent: none; 
- pure substance added to water
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISMS
- Post-hatch transfer time: < 24 hours
- Age: < 24 hours
- Feeding: Artimia salina nauplii (sufficient, some not eaten), three  times daily, on weekends only two times daily
- Feeding during test: After 1 week additionally 5 g trout starter/day
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Hardness:
- 47-48 mg/l as  CaCO3
Test temperature:
- 24.2-25.6 (mean 25.1) °C
pH:
- pH: 7.5-7.8
Nominal and measured concentrations:
- Concentrations: 5 per test (2.14 / 4.18 / 8.29 / 15.61 / 22.66 mg/l and  2.18 / 4.15 / 8.78 / 14.51 / 27.63 mg/l)
Details on test conditions:
TEST SYSTEM
- Test type: flow-through

- Renewal of test solution: 25 fold in 24 hours
- Exposure vessel type: days 1-4: glass embryo cup; then release to glass  vessel 46 cm x 16 cm x 18 cm, filled with 10 cm deep water = ca. 8.25 l; 
- Number of replicates, individuals per replicate: 2, 15-35
- Test temperature: measured twice weekly
- Dissolved oxygen: once/week, always > 90 %
- pH/hardness/alkalinity: twice during test
- alkalinity 38-40 mg/l as CaCO3
- Controls: dilution water

DILUTION WATER
- Source: sand filtered Lake Superior water
- Alkalinity: 40-42 mg/l as CaCO3
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
4.2 - 15.6 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
8.8 - 22.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight

Test 1: NOEC (weight) = 15.6 mg/l
Test 2: NOEC (weight) = 4.2 mg/l


Geometrical mean test 1 and 2: NOEC (weight) = 8.1 mg/l
Feeding was identified to be crucial for the test. The total growth of fish


was higher when the test was started early during the week (monday / tuesday) 


because then fish were fed three times daily during the first days.

Executive summary:

Two flow-through tests using Pimephales promelas (Lemke et al., 1983) were conducted according to a method proposed by the U.S. EPA. After 32 days of exposure, the following endpoints were obtained:

Test 1: NOEC (weight) 15.6 mg/l)

Test 2: NOEC (weight) 4.2 mg/L

The authors explained the deviation of the results with differences in the feeding regime. The study was assessed as "reliable with restrictions".

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Method: other: Early Life Stage Test
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
MONITORING OF TEST SUBSTANCE CONCENTRATION: 
- twice weekly
Vehicle:
no
Details on test solutions:
STOCK AND TEST SOLUTION AND THEIR PREPARATION
- Dispersion: Diluter
- Vehicle, solvent: none
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISMS
- Age/size/weight/loading: embryos <= 48 hours old
- Pretreatment: eggs from spawning substrates; dead and unfertilized eggs  were discarded
- Feeding during test: live, newly-hatched brine shrimp at least twice  daily on weekdays and once daily on weekends
- Controls: freshwater
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Hardness:
- Hardness: 35 (24-70) mg/l CaCO3
Test temperature:
- Test temperature: 24.5 +/- 1 °C
pH:
- pH: 7.4 (7.1-7.6)
Dissolved oxygen:
- Oxygen content: 7.9 (6.2-8.4) mg/l
Nominal and measured concentrations:
- Measured concentrations: 112 +/- 15; 56 +/-13; 30 +/- 11; 19 +/- 9; 11 +/- 6 mg/l
Details on test conditions:
TEST SYSTEM

- Renewal of test solution: 88 volume additions in 24 hr
- Exposure vessel type: 1 l glass beakers, 900 ml solution, 10.5 cm deep,  5.7 cm square embryo scren trays
- Number of replicates, fish per replicate: 4 replicates, about 8 embryos  per chamber
- Intensity of irradiation: approximately 270 lux on surface
- Photoperiod: 16 h light / 8 h dark

DILUTION WATER
- Source: Western Fish Toxicology Station, Corvallis, Oregon
- Alkalinity: 28 (20-47) mg/l CaCO3

STATISTICAL METHODS:
- two-way analysis of variance (ANOVA) after arcsin transformation (for percentages) or conversion to logs (for  weights and lengths); 
- Williams multiple comparison test for differences among treatments (P = 0.05)
Duration:
35 d
Dose descriptor:
other: LLC
Effect conc.:
112 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
11 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
19 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight

Survival:      LOEC = 112 mg/l; NOEC = 56 mg/l
Fork length:   LOEC =  30 mg/l; NOEC = 19 mg/l
Weight:        LOEC =  19 mg/l; NOEC = 11 mg/l
Geometric mean NOEC (fork length + weight) =  14.5 mg/l

Executive summary:

A flow-through test using Pimephales promelas embryos (maximum 48 hours old) was conducted by Cairns and Nebeker (1982). The fish were exposed over a period of 35 days to 5 concentrations in well water and one control. The test indicated that survival was affected by 112 mg/l but not by 56 mg/l and lower. Observing the fork length, a NOEC of 19 mg/l and a LOEC of 30 mg/l were obtained, while the fish weight was affected by 19 mg/l and higher but not by11 mg/l. Based on fish weight as the most sensitive endpoint the NOEC was determined as 11 mg/l. The main endpoints are as follows:


 


NOECweight: 11 mg/L


LOWCweight: 19 mg/L


 


The study was assessed as "reliable with restrictions".

Description of key information

For the most sensitive species, Pimephales promelas, NOECs from three early life stage tests are available (Cairns and Nebeker, 1982; Lemke et al.,
1983, test 1+2, exposure period 32-35 days) that are in the same order of magnitude. Therefore, the geometric mean of these 3 NOECs is calculated resulting in a value of 8.9 mg/l. The studies were conducted according to accepted scientific principles and were assessed as "reliable with
restrictions".

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
8.9 mg/L

Additional information

A flow-through test using Pimephales promelas embryos (maximum 48 hours old) was conducted by Cairns and Nebeker (1982). Observing the fork length, a NOEC of 19 mg/l and a LOEC of 30 mg/l were obtained, while the fish weight was affected by 19 mg/l and higher but not by 11 mg/l. Based on fish weight as the most sensitive endpoint the NOEC is determined as 11 mg/l. Similar results were obtained in 2 flow-through tests using the same species (Lemke et al., 1983). After 32 days of exposure, NOECs for the endpoint fish weight of 4.2 (2nd test) and 15.6 mg/l (1st test) were obtained. The authors explained the deviation of the results with differences in the feeding regime.