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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP study according to protocol
Qualifier:
according to
Guideline:
other: FIFRA guideline 72-4 ELS test with Fathead minnow
Deviations:
yes
Remarks:
Temperature was not within the criteria of the guideline during the entire study.
Qualifier:
according to
Guideline:
other: Draft OECD : Fish toxicity test on egg and sac-fry stages
Principles of method if other than guideline:
The effect of Halamid on eggs and larvea of fathead minnow was studied during a period of 35 days in a flow-through system. Survival, wet-weight and fish length were determined. 
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The control and the high, middle, and low test concentrations were sampled and analyzed for Chloramine-T concentration prior to the start of the definitive exposure. During the in-life phase of the definitive study, water samples were removed from both replicate test solutions of each treatment level and the control on test days 0, 5, 12, 14, 28, 33 and 35 for analysis of Chloramine-T concentration. Additional sampling and analysis of the exposure solution in the delivery tubing of the high and low exposure solutions (A,B replicates) was also conducted at day 0, 5, 14, 28, 33 and 35. Three Quality Control (QC) samples were also prepared at each sampling interval and remained with the set of exposure solution samples throughout the analytical process. The results of these analyses (QC) were used to judge the precision and quality control maintained during the analysis of exposure solution samples.
Vehicle:
no
Details on test solutions:
Stock solutions (18 mg A.l./mL) were prepared on test days -1, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35 by diluting approximately 1.763 g (1.796 g of active ingredient) of Chloramine-T with distilled water to a total volume of 100 mL. The resulting stock solutions were observed to be clear and colorless. An aliquot of the 18 mg A.l./mL stock solution was analyzed on test day 12 and reanalyzed on test day 19. The purpose of these analyses was to demonstrate the stability of the stock solution over seven days.

A 50-mL gas-tight Glencom syringe in conjunction with a Sage syringe pump (Model 355) was mechanically activated to deliver 0.0167 mL/minute of the 18 mg A.l./mL stock solution to the exposure system's chemical mixing cell. Dilution water was continuously delivered to the mixing cell at a rate of 0.200 L/minute. This delivery rate (test material and dilution water) provided a solution containing a Chloramine-T concentration equal to the highest treatment level (1.5 mg A.I./L), which was then diluted (50%) to provide the remaining nominal concentrations (0.047, 0.094, 0.1 9, 0.38 and 0.75 mg A.I./L).
Test organisms (species):
Pimephales promelas
Details on test organisms:
- Source: Springborn brood stock, Wareham, Massachusetts, USA
- Wild caught: No
- Age/size: At test initiation the embryos were ≤ 24 hours old
- Kind of food: The fry were fed brine shrimp nauplii. Representative samples were analyzed for the presence of pesticides, PCBs and toxic metals. Food sources were considered to be of acceptable quality since the total concentration of pesticides measured was less than 0.3 mg/kg
- Amount of food: The fry were fed an excess of live brine shrimp ad libitum to make sure all fry had access to equal amounts of food
- Feeding frequency: Three times daily on weekdays and twice daily on weekends
- Post-hatch transfer time: Unknown
- Time to first feeding: After completion of the hatch, 5 days, the fry were fed
- Feeding of animals during test: Yes, see above
- Treatment for disease within 2 weeks preceding test: No
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Remarks on exposure duration:
(post hatch)
Post exposure observation period:
Fathead minnow eggs were obtained from brood stock maintained at Springborn Laboratories, Inc. On the day prior to test initiation, spawning tiles were placed in the fathead minnow brood culture unit. The following day, the eggs were collected from the tiles and impartially distributed to the egg incubation cups in the following manner: fourteen unlabeled, unassigned petri dishes were set in a shallow waterbath maintained at 25 °C. A small amount of water from the control aquaria of the exposure system was placed in each dish.
Hardness:
23 - 32 mg/l (CaCO3)
Test temperature:
24 - 25 °C
pH:
6.8 - 7.4
Dissolved oxygen:
7.8 - 8.1 mg/l
Nominal and measured concentrations:
- Nominal: 0.047-0.054-0.11-0.24-0.51 and 1.1 mg/l
- Measured: 0.039-0.054-0.11-0.24-0.51-1.1 mg/l (average recovery of 81.6 of the nominal values)
Details on test conditions:
DILUTION WATER
- Source: Town of Wareham untreated well water 
- Aeration: yes
- Alkalinity: 20 - 23 mg/l
- TOC: 1.1 mg/l
- TSS: not described
- Conductance: 110 - 130 micromhos/cm 
- Holding water: identical 


TEST SYSTEM
- Test type: flow-through 
- Dosing rate: continuous
- Renewal of test solution: 6.5 aquarium volumes per 24 hours, 90% replacement time 8.0 hours.
- Exposure vessel type: 11 L test aquaria were used in which embryo incubation cups were placed (glass jars, 5 cm O.D., 8 cm high with 40-mesh Nitex® bottom)  
- Number of replicates: 2 replicates 
- Number of animals/vessel: 60 embryos per incubation cup during the hatching period and 40 larvae per aquarium during the post-hatching period
- Test temperature: 25 +/- 1 degree C
- Adjustment of pH: no
- Intensity of irradiation: 540-970 lux
- Photoperiod: 16:8 dark/light cycle


ENDPOINTS ASSESSED: mortality, wet-weight, length 
Reference substance (positive control):
no
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
1.5 mg/L
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
2 mg/L
Details on results:
RANGE FINDING TEST 
no mortality at 0.5 and 1.0 mg/l, but 50% at 2.0 mg/l

CHEMICAL ANALYSES
Analysis of exposure solutions during the pretest period resulted in measured concentrations which averaged 81.6% of the nominal concentrations.
The concentrations in the solutions being delivered to the exposure vessels were generally equal to the nominal concentrations. Therefore it was concluded that the appropriate concentrations were delivered to the exposure vessels.
The exposure solutions at the highest treatment level showed a slight reduction during the course of the study; during the initial week the measured concentration was approximately 100% of the nominal and from day 12-35 of the test 71% of the nominal level. At lower exposure levels was in general agreement during the first week of exposure but the concentrations varied much more with time.

BIOLOGICAL OBSERVATIONS
At the completion of the hatching period (day 5), survival of organisms exposed to the control solution averaged 86%. Fathead minnow survival at the completion of the hatching period (day 5) in the treatment level solutions (0.039 to 1 .I mg A.I./L) ranged from 83 to 89%.

Following 30-days post-hatch exposure (test day 35), larval survival in the control solution averaged 80%. During this same period, larval survival in the treatment levels tested ranged from 85 to 94%.

At test termination, total length of larvae in the control solution averaged 31.5 mm. The total length of larvae exposed to the treatment level solutions (0.039 to 1.1 mg A.I./L) ranged from 31.2 to 31.7 mm.
Similarly, the wet weight of organisms in the control solution averaged 0.31 9 g. The wet weight of larvae exposed to the concentration range tested (0.039 to 1.1 mg A.I./L) ranged from 0.307 to 0.330 g.
Reported statistics and error estimates:
significance of differences were determined using William's test (data distribution checked using Bartlet's test and Shapiro Wilks test). End-points were compared to controls.
METHOD OF CALCULATION: Survival and hatching was compared to control and differences were statistically determined.

Survival, length and wet weight

Mean measured concentration (mg a.i./L)

Replicate

Organism survival at hatch (%)

Larval survival at test termination (%)

Total length (mm)

Wet weight (g)

control

A

87

83

30.7 (2.54)

0.294 (0.071)

B

85

78

32.3 (2.34)

0.345 (0.070)

Mean

86

80

31.5 (2.56)

0.319 (0.074)

0.039

A

88

90

31.2 (4.48)

0.310 (0.100)

B

78

85

31.4 (2.65)

0.313 (0.089)

Mean

83

88

31.3 (3.68)

0.311 (0.094)

0.054

A

90

85

32.3 (2.02)

0.332 (0.073)

B

88

85

31.1 (3.97)

0.327 (0.086)

Mean

89

85

31.7 (3.18)

0.330 (0.080)

0.11

A

72

90

30.7 (2.53)

0.288 (0.068)

B

93

88

32.1 (1.31)

0.326 (0.051)

Mean

83

89

31.4 (2.14)

0.307 (0.063)

0.24

A

88

93

31.3 (3.67)

0.325 (0.088)

B

90

90

31.3 (2.17)

0.322 (0.067)

Mean

89

91

31.3 (3.01)

0.324 (0.078)

0.51

A

88

93

31.8 (2.25)

0.334 (0.079)

B

90

93

30.6 (4.80)

0.310 (0.109)

Mean

89

93

31.2 (3.77)

0.322 (0.095)

1.1

A

87

95

31.8 (3.06)

0.332 (0.085)

B

88

93

31.2 (1.92)

0.319 (0.067)

Mean

88

94

31.5 (2.56)

0.325 (0.077)


Validity criteria fulfilled:
yes
Conclusions:
The highest test concentration was once below 80% (on day 19) on all other days the measured concentrations were within 80-120%. No effect was observed and therefore the variation in the measured concentrations did not affect the outcome of the test and the test is considered valid. No adverse effects were observed at the highest concentration tested in the definitive test. Therefore, the NOEC is 1.5 mg/L.
Executive summary:

PROTOCOL TITLE: "Protocol for Conducting an Early Life Stage Toxicity Test with Fathead Minnow (Pimephalespromelas) Following FIFRA Guideline 72-4 and Draft OECD Guideline entitled 'Fish Toxicity Test on Egg and Sac-Fry Stages"', Springborn Laboratories' Protocol #:100792/FIFRA-OECD and Protocol Amendment # I dated 8 January 1993 (Appendix I). The methods described in this protocol follow the procedures described in FIFRA Guideline 72-4 (U.S. EPA, 1982) and draft OECD guidelines (OECD, 1992).

REPORT NUMBER: 93-9-4927

STUDY NUMBER: 11784.1092.6103.120

TEST SUBSTANCE: Chloramine-T - Lot# 02992045201 39, CAS # I27-65-1

DATE RECEIVED: 20 November 1992 and 31 December 1992

DESCRIPTION: A white powder; 101.9% active ingredient based on a Certificate of Analysis

EXPERIMENTAL START DATE: 20 May 1993

EXPERIMENTAL TERMINATION DATE: 24 June 1993

TEST ORGANISM: Pimephales promelas, source - Springborn brood stock

TEST CONDITIONS: 35 day (30 day post-hatch) duration, 22 to 25 OC, illumination of 16 hours light:8 hours dark with an illumination of 50 to 90 footcandles (540 to 970 lux) measured at test initiation

DILUTION WATER: Well water supplemented with Town of well water

pH: 6.9 to 7.2

Specific conductivity: 1 10 to 130 µmhos/cm

Total hardness as CaCO3: 23 to 32 mg/L

Total alkalinity as CaCO3: 20 to 23 mg/L

NOMINAL TEST CONCENTRATIONS: 0.047, 0.094, 0.19, 0.38, 0.75 and 1.5 mg A.I./L

MEAN MEASURED CONCENTRATIONS: 0.039, 0.054, 0.11, 0.24, 0.51 and 1.1 mg A.I./L

RESULTS: The absence of any significant biological effects during the definitive exposure established the No-Observed-Effect Concentration (NOEC) as 1.1 mg A.I./L, the highest measured concentration of Chloramine-T tested. The Lowest-Observed-Effect Concentration (LOEC) was determined by evaluating the data obtained during the 12-day preliminary study and was established as 2.0 mg A.I./L (nominal).

Description of key information

An evaluation on the effect of Halamid on embryos and larvae of fathead minnow were studied in accordance to FIFRA Guideline 72-4 and Draft OECD Guideline entitled 'Fish Toxicity Test on Egg and Sac-Fry Stages". Embryo and sac-fry stages of fish were exposed to a control and to Halamid concentrations of 0.047, 0.094, 0.19, 0.38, 0.75 and 1.5 mg A.I./L in a flow-through system. The total exposure period was 35 days and samples were analyzed by direct aqueous injection on a HPLC system using UV detection. Survival, wet-weight and fish length were determined. Measured concentrations were within 80-120%, with the exception of the highest test concentration which fell below 80% (on day 19). No effect was observed and therefore the variation in the measured concentrations did not affect the outcome of the test and the test is considered valid.The absence of any significant biological effects during the definitive exposure established the No-Observed-Effect Concentration (NOEC) as 1.1 mg A.I./L, the highest measured concentration of Chloramine-T tested. The Lowest-Observed-Effect Concentration (LOEC) was determined by evaluating the data obtained during the 12-day preliminary study and was established as 2.0 mg A.I./L (nominal).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
1.1 mg/L

Additional information