Bis(tributyltin) oxide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13th September 1989 - 2nd April 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Experiment performed under GLP conditions, but not according to standardised guidelines. Analytical verification of test concentrations performed, but resulted in a low recovery of nominal concentrations

Qualifier:

no guideline followed

Principles of method if other than guideline:

A flow-through saltwater life-cycle toxicity lest was conducted at the Gulf Coast Research Laboratory in Ocean Springs, Mississippi, to determine the chronic effect of bis(tributy!tin) oxide (TBTO) to the sheepshead minnow (Cyprinodon variegcaus Lacepede). The 180 day study was initiated on October 4, 1989 with embryos isolated from culture and concluded on April 2, 1990. The criteria of effect for the parental generation included embryo survival; survival and growth of juveniles after 30, 61, and 90 days exposure; and survival, reproduction, and growth of adults. Progeny generation effects included embryo survival, and juvenile survival and growth 30 days post isolation. Results of the test are reported as the no observed effect concentration (NOEC), the lowest observed effect concentration (LOEC), and the final chronic value (the geometric mean of the NOEC and LOEC). Chemical analysis of all test media utilized n-pentyl derivatization followed by gas chromatography/flame photometric detection.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.25, 0.5, I.O, 2.0, and 4.0 µg (Sn)/L
- Sampling method: 200mL collected from each concentration weekly (once a week minimum) from below the water surface in an area not in contact with the chamber walls.
- Sample storage conditions before analysis: Analysed immediately or acidified and frozen in teflon spearatory funnels

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Study Stocks were prepared by adding 0.5 milliliter (ml) of TBTO to 45 L of distilled water and stirring with a teflon-coated stir bar for a minimum of 4 days.
- Eluate: Distilled water + TBTO
- Differential loading: 0.195 g/L
- Controls: Water only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc):N/A

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow (Cyprinodon variegatus)
- Source: In-house laboratory culture
- Age at study initiation (mean and range, SD): Embryos less than 24 hours old
- Method of breeding: Adult spawners maintained in static recirculating system
- Feeding during test
- Food type and frequency: While maturing, during reproduction and to termination, fish were fed frozen adult brine shrimp (San Francisco Bay Brand Frozen Brine Shrimp, Newark, California) once daily and commercial flake food (Stress Flakes, Novalek, Hayward, California) once daily.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

180 d

Hardness:

Not reported

Test temperature:

25 to 32 °C

pH:

7.8 to 8.4

Dissolved oxygen:

≥ 4.4 mg/L (≥ 63% saturation)

Salinity:

15 ± 1 ppt

Nominal and measured concentrations:

Measured (Nominal) Concentrations: N.D. (Control), 0.17 (0.25), 0.27 (0.5), 0.55 (1.0), 1.32 (2,0), and 2.20 (4.0) µg (Sn)/L

Details on test conditions:

TEST SYSTEM
- Emybro cups : 150 mm petri dish bottoms with a 15 cm tall 40-mesh nylon collar
- Test vessel: Glass aquaria (90 x 45 x 26 cm)
- Type: open
- Material, size, headspace, fill volume: Glass
- Aeration: Via flow-through system
- Type of flow-through: Proportional diluter
- Renewal rate of test solution : During the 180 day study the diluter cycled 60,940 times, providing a flow rate to each aquarium of approximately 14 cycles per hour. This flow rate was sufficient to provide a 95 percent daily volume addition and to allow a maximum loading density of 0.195 g/L during the exposure.
- No. of fertilized eggs/embryos per vessel: 50
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 0.195 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Salt water used for culture and testing was filtered (10-µm) natural seawater collected from the Santa Rosa Sound near Pensacola, Florida. The only exception was during the final week of the study when water was collected from the Gulfarium in Fort Walton Beach, Florida. This second source was utilized when the ambient salinity of the primary source fell below 15 ppt. The dilution water was adjusted to a salinity of approximately 15 ppt with artesian well water from the GCRL facility in Ocean Springs, Mississippi.
- Total organic carbon: 1.12 to 4.33 mg/L
- Particulate matter: 79 to 93 ppm
- Metals: Below or at detection limits
- Pesticides: Below detection limits
- Salinity: 15 ± 1 ppt
- Culture medium different from test medium: No
- Intervals of water quality measurement: Weekly

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light: 8 hours dark
- Light intensity: 473 ± 54 Lux

EFFECT PARAMETERS MEASURED: Survival, growth, reproduction, progeny

Reference substance (positive control):

no

Duration:

180 d

Dose descriptor:

NOEC

Effect conc.:

0.17 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

other: Survival, growth, reproduction endpoints

Duration:

180 d

Dose descriptor:

LOEC

Effect conc.:

0.27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Survival, growth, reproduction endpoints

Duration:

180 d

Dose descriptor:

other: Final Chronic Value

Effect conc.:

0.21 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

adult mortality

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: See table below
- Days to hatch or time to release of young: No significant effect of treatment
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: No significant effect of treatment
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): Not reported
- Observations on body length and weight of young and/or exposed parents at one or more time periods: No significant effect of treatment
- Number of healthy fish at end of test:
- Detailed data on spawning, egg numbers, fertility, and fecundity: No significant effect of treatment
- Effect concentrations exceeding solubility of substance in test medium: None
- Incidents in the course of the test which might have influenced the results: Malfunctioning diluter systems

Reported statistics and error estimates:

Since each control and treatment concentration was represented by duplicate aquaria, continuous data (growth and fecundity) was analyzed by a hierarchical or nested analysis of variance (Steel and Torrie, 1960; Zar, 1974). When the F value for the treatment effect was significant, all exposure concentration responses were compared with the control response by using a two-tailed Dunnett's test (Zar, 1974; Dunnett, 1955).
Dichotomous data (embryo, juveniie, and parental survival) were analyzed using 2x2 contingency tabes. A two-tailed Fisher's exact lest (Zar, 1974) was used to determine differences between replicates of each treatment group. Replicate responses were then pooled and each treatment compared to the control using chi-square analysis of contingency tables (Zar, 1974). The significance level used in all statistical tests was 0.05.

Table 1: Survival of parental generation during a 180 day test

Concentration (ppb)	Percent hatch	% Fry survival	% 61-day survival	% 90-day survival	% 120-day survival	% 145-day survival	% 163-day survival
Control	85	85.9	94	94	92	90	82
0.17	85	94.1	98	98	94	86	82
0.27	85	95.3	94	88	74*	50*	34*
0.55	86	94.2	86	84	78	54*	40*
1.32	80	57.5*	83	41*	11*	0*	0*
2.20	67*	0*	--	--	--

* significantly different from controls

Validity criteria fulfilled:

not applicable

Conclusions:

Embryo survival of parental generation sheepshead minnows (Cyprinodon variegatus) was significantly reduced by TBTO at a concentration of 2.20 µg (Sn)/L [5.38 µg TBT/L]. Survival of fry and juveniles was significantly affected at concentrations ≥1.32 µg (Sn)/L [3.22 µg TBT+/L].
Effect to parental generation survival increased to include all concentrations ≥ 0.27 µg (Sn)/L [0.66 µg TBT+/L] by day 145 of the exposure. Growth as Standard length of parental generation sheepshead minnows was significantly reduced on day 90 at 1.32 µg (Sn)/L [3.22 µg TBT/L]. However, there were no significant reductions in length at day 30 or to adults when terminated. Wet and dry weights of the parental generation as juveniles on day 30 were not significantly different across treatments and controls. Wet weights of second spawning trial females were significantly less than controls in the 0.17 and 0.55 µg (Sn)/L [0.42 and 1.34 µg TBT/L] concentrations, but were not in the 0.27 µg (Sn)/L [0.66 µg TBT/L] concentration. Furthermore, significant growth differences were not observed in second spawning trial male fish, or in either males or females from the first spawning trial or at day 163.
Fecundity as measured by the average number of viable eggs produced per female per day was not significantly reduced at concentrations ≤1.32 µg (Sn)/L [3.22 µg TBT/L]. Survival of progeny embryos from treatment chambers was significantly affected by 1.32 µg (Sn)/L [3.22 µg TBT/L].
Survival and growth of juveniles from the progeny generation were not significantly affected at concentrations ü 0.55 µg (Sn)/L [1.34 µg (TBT/L] when isolated into unamended dilution water or the treatment of origin. The effect of bis(tributyltin) oxide as observed in this study to the survival of parental generation sheepshead minnow establishes the LOEC as 0.27 µg (Sn)/L [0.66 µg TBT/L], the NOEC as 0.17 µg (Sn)/L [0.42 µg TBT/L] and the Final Chronic Value as 0.21 µg (Sn)/L [0.51 µg TBT/L].

Executive summary:

SUMMARY

The Consortium of Tributyltin Manufacturers, Atochem North America, Incorporated of King of Prussia, PA and Sherex Chemical Company, Incorporated of Dublin, OH

Study Director: William W. Walker

Location of Study: Gulf Coast Research Laboratory, Ocean Springs, MS

Location of Raw Data and Final Report: Gulf Coast Research Laboratory, Ocean Springs, MS

Test Substance: Bis(tributyltin) oxide; CAS No. 56-35-9; 97.5 percent bis(tributyltin) oxide (39.3 percent elemental tin) containing 0.089 percent dibutyltin oxide (DBTO), 0.09 percent Chloride, and 2.34 percent other tin compounds as reported by M&T Chemicals, Inc. An analysis for purity on September 11, 1989 by the Analytical Chemistry Section of GCRL indicated 97.9 percent bis(tributyltin) oxide, 1.0 percent dibutyltin dichloride and 1.05 percent tetrabutyltin.

Subject: Final Report, Life-Cycle Saltwater Fish Test

Measured (Nominal) Concentrations: N.D. (Control), 0.17 (0.25), 0.27 (0.5), 0.55 (1.0), 1.32 (2,0), and 2.20 (4.0) /µg (Sn)/L

Test Dates: Range finding: September 13 to 29, 1989

Definitive: October 4, 1989 to April 2, 1990

Length of Study: 180 days

Results: The NOEC was 0.17 µg (Sn)/L [0.42 /µg (TBT+)/L]; the LOEC was 0.27 µg (Sn)/L [0.66 µg (TBT+)/L]; and the Final Chronic Value was 0.21 /µg (Sn)/L [0.51 µg

(TBT+)/L] based on survival of parental generation sheepshead minnow.

Test Species: Sheepshead Minnow {Cyprinodon variegatus Lacepede)

Source of Organisms: Gulf Coast Research Laboratory, Ocean Springs, MS

Dilution Water: Natural Filtered Seawater diluted with well water to 15 ± 1 ppt salinity

Water Quality: Test temperature ranged from 25 to 32°C; dissolved oxygen remained >/- 4.4 mg/L (>/- 63 percent)

Description of key information

Sheepsheads minnows (Cyprinodon variegatus Lacepede) were exposed to bis (tributyltin) oxide in a flow-through system for 180 days in a life cycle toxicity study.

Embryo survival of parental generation sheepshead minnows (Cyprinodon variegatus) was significantly reduced at 2.20 µg (Sn)/L [5.38 µg TBT/L]. Survival of fry and juveniles was significantly affected at >1.32 µg (Sn)/L [3.22 µg TBT/L].

Effect to parental generation survival increased >0.27 µg (Sn)/L [0.66 µg TBT/L] by day 145 of the exposure. Growth (standard length) of parental generation was significantly reduced on day 90 at 1.32 µg (Sn)/L [3.22 µg TBT/L]. There were no significant reductions in length at day 30 or to adults at termination. Wet and dry weights of the parental generation as juveniles on day 30 were not significantly different across treatments and controls. Wet weights of second spawning trial females were significantly less than controls in the 0.17 and 0.55 µg (Sn)/L [0.42 and 1.34 µg TBT/L] concentrations, but not in the 0.27 µg (Sn)/L [0.66 µg TBT/L] concentration. Furthermore, significant growth differences were not observed in second spawning trial male fish, or in either males or females from the first spawning trial or at day 163.

Fecundity as measured by the average number of viable eggs produced per female per day was not significantly reduced at <1.32 µg (Sn)/L [3.22 µg TBT/L]. Survival of progeny embryos from treatment chambers was significantly affected by 1.32 µg (Sn)/L [3.22 µg TBT/L].

Survival and growth of juveniles from the progeny generation were not significantly affected at concentrations 5 0.55 µg (Sn)/L [1.34 µg TBT/L] when isolated into unamended dilution water or the treatment of origin. The effect of bis(tributyltin) oxide as observed in this study to the survival of parental generation sheepshead minnow establishes the LOEC as 0.27 µg (Sn)/L [0.66 µg TBT/L], the NOEC as 0.17 µg (Sn)/L [0.42 µg TBT/L] and the Final Chronic Value as 0.21 µg (Sn)/L [0.51 µg TBT/L]

Key value for chemical safety assessment

Marine water fish

Marine water fish

Effect concentration:

0.17 µg/L

Additional information

The key study Walker, W.W. (1991), was in compliance with GLP, but was not conducted to a standardised guideline. The study is considered reliable with reasonable restrictions and was assigned a reliability score of 2.