Glycerol trinitrate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 1987-March 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets National standards method with acceptable resctrictions.

Qualifier:

according to guideline

Guideline:

other: Draft No.10 of the ASTM proposed guide for conducting early life-stage toxicity tests with fish (Goodman, 1986)

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- The stability of the NG stock solution were periodically checked during storage to assure that no decomposition occured. Standard solutions of NG were prepared by dilution of the stock solution freshly prepared each day of analysis. Standard solutions with concentrations of 10.0, 5.0, 2.5, and 1.0 µl/L were used.- Aqueous samples from bioassays were injected directly into HPLC after filtration to remove particles > 0,45 µm.In the cases where samples could not be analysed immediately following filtration, the filtered samples were stored at 4°C in amber glass vials fitted with Teflon-lined caps and analyzedwithin 24 h from the time the samples were originally taken from the test aquaria.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION - Basic solution (the shipment): 1 g NG dissolved in JHU/APL well water. NG solutions were keep in the dark at 4°C in glass bottles.- NG stock solutions were prepared by adding appropriate amounts of the material to aerated JHU/APL well water and stirred for 4 to 8 h. No NG stock solutions were heated during preparation or filtering at 0.45 µm. All stock solutions were prepared in amber glass containers.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM- Common name: Fathead minnow- Source: the JHU/APL culture. The JHU/APL culture was initiated with mature fathead minnows obtained from the U.S.EPA Environmental Monitoring and Support Laboratory-Cincinnati, Ohio.- Age at study initiation (mean and range, SD): embryo (< 12h)- Method of breeding: Spawning fish were cultured in fiber glass tanks containing JHU/APL well water held at 25 +/- 1 °C. The spawning adults were fed a diet of frozen brine shrimp (Artemia sp.) and TetraMin® staple food. twice daily.Excess food was removed daily. Five sets of spawning fathead minnows were maintained in the culture tanks at ration of 1 male : 3 females. Replacement spawners were rotated at approx. 3-month intervals. Fathead minnow embryos were collected on spawning substrates. Fry were reared on brine shrimp nauplii in 19 L aquaria at 25 +/- 1°C in JHU/APL well water.POST-HATCH FEEDING- Type/source of feed: live brine shrimp (< 24h old)- Frequency of feeding: beginning at day 2 post-hatch, three times daily on weekdays and twice daily on weekends

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

28 d

Hardness:

225 (190-250) mg/L as CaCO3

Test temperature:

24.9 (24.1-25.5) °C

pH:

7.5 (6.8-8.1)

Dissolved oxygen:

7.5 (7.1-7.8) mg/L

Nominal and measured concentrations:

Nominal concentration: 0.13, 0.22, 0.36, 0.6, 1 mg/LMeasured (mean) concentration): 0.12, 0.2, 0.33, 0.57, 0.94 mg/L

Details on test conditions:

TEST SYSTEM- Embryo cups (if used, type/material, size, fill volume): 0.2-L glass cylinder, fill volume: ≈ 0.14 L- Test vessel: 10-L glass aquarium- Fill volume: 6.4 L - Volume additions per day: 5.3 L (mean volume addition per day for the complete exposure period)- Type of flow-through: solenoid-activated proportional dilutor systems- No. of fertilized eggs/embryos per vessel: 40 embryos per cup- No. of vessels per concentration (replicates): 2- No. of vessels per control (replicates): 2- Biomass loading rate: 0.365 g/L (mean loading (g wet weight/L) at the end of the exposure period for control organismsTEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Non-chlorinated deep well located at The Johns Hopkins University Applied Physics Laboratory (JHU/APL) in Shady Side, MD.- Total organic carbon: 19 mg/L- Metals: Sb, As, Be, Pb, se, Tl < 0.005 mg/L, Cd < 0.001 mg/L, Cr < 0.05 mg/L, Cu < 0.02 mg/L, Hg < 0.0002 mg/L, Ni < 0.2 mg/L, Ag <0.01 mg/L, Zn < 0.04 mg/L- Alkalinity: 110 (75-140) mg CaCO3/L- Conductivity: 342 (300-390) µmhos/cm- The water used in was analyzed for 43 Priority Pollutants, eight nonpriority, but "Hazardous" substances, 26 pesticides, and 13 metals. None of them were detected with detection limits of 2, 2, 0.1 µg/, and <0.2 - <0.0002 mg/, respectively.- Intervals of water quality measurement: A comprehensive chemical analysis of the well water was conducted two times during the study. The analyses were separated by approx. 18 months.OTHER TEST CONDITIONS- Photoperiod: 16-h light:8-h dark- Light intensity: fluorescent lights, 60-85 foot candles at the surface of the culture vesselsEFFECT PARAMETERS MEASURED: hatching success (after 4 days of exposure), survival of larvae (after 28 days of post-hatch exposure), growth of larvae (after 28 days of post-hatch exposure)POST-HATCH DETAILS- post-hatch fish were observed daily for developmental abnormalities and mortality- total lenght, blotted wet weight and dry weight were determine for all fish from each treatment

Duration:

4 d

Dose descriptor:

LOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: percent hatch

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: percent hatch

Details on results:

- 100% mortality of larvae occured in treatment groups with concentartion of NG 0.57 and 0.94 mg/L.- A total of 10 fish with spinal deformities were observed during the study. Five of the 10 fish died before the 28-d exposure was completed. The deformities occurred at 0.33 and 0.2 mg/L, one of the 10 deformed fish was a control fish.

Reported statistics and error estimates:

Arc-sine square root trasformations were made on survival and hatching success raw data before further data analyses were performed.Both transformed and raw data were then subjected to a chi-square test of normaliy and Bartlett´s test for homogeneity of variance. When the data sets met the assumptions of normality and homogeneity of variance, a parametric statistic was used. Dunnett´s test was used when the number of replicates was constant among treatments. Duncan´s new multiple range test was also occasionally used.A minimum probability level of 0.05 was used for all tests.

Conclusions:

NG caused a significant reduction in hatching success of the enbryos after 4 d of exposure at a concentration 0.2 mg/L. 0.12 mg/L did not affect hatching success. Larval survival after 28 d of exposure was reduced at 0.33 mg/L, while no effect occurred at 0.2 mg/L. Total lenght, wet weight and dry weight were all reduced at 0.33 mg/L, but not at 0.2 mg/L. The LOEC and NOEC based on hatchingsuccess are 0.2 and 0.12 mg/L, respectively.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 1987-March 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets National standards method with acceptable resctrictions.

Qualifier:

according to guideline

Guideline:

other: Draft No.10 of the ASTM proposed guide for conducting early life-stage toxicity tests with fish (Goodman, 1986)

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- The stability of the NG stock solution were periodically checked during storage to assure that no decomposition occured. Standard solutions of NG were prepared by dilution of the stock solution freshly prepared each day of analysis. Standard solutions with concentrations of 10.0, 5.0, 2.5, and 1.0 µl/L were used.- Aqueous samples from bioassays were injected directly into HPLC after filtration to remove particles > 0,45 µm.In the cases where samples could not be analysed immediately following filtration, the filtered samples were stored at 4°C in amber glass vials fitted with Teflon-lined caps and analyzedwithin 24 h from the time the samples were originally taken from the test aquaria.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION - Basic solution (the shipment): 1 g NG dissolved in JHU/APL well water. NG solutions were keep in the dark at 4°C in glass bottles.- NG stock solutions were prepared by adding appropriate amounts of the material to aerated JHU/APL well water and stirred for 4 to 8 h. No NG stock solutions were heated during preparation or filtering at 0.45 µm. All stock solutions were prepared in amber glass containers.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM- Common name: Rainbow trout- Source: Pennsylvania Fish Commission, Benner Spring Fish Research Station, State College, PA- Age at study initiation: eyed embryo (12-14 d)ACCLIMATIZATION:- Eyed embryos were gradually adjusted to JHU/APL well water at 12 +/- 1°C over 24-h period and held an additional 24 h prior to testing. The eyed embryos were held in reduced light of < 25 foot candles at the surface of the water.POST-HATCH FEEDING- Type/source of feed: ground Salmon Starter Meal- Amount given: the amount of ration per feeding was approx. 4% dry food/wet weight fish- Frequency of feeding: three times daily on weekdays and twice daily on weekends

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

60 d

Hardness:

165 (132-192) mg/L as CaCO3

Test temperature:

12.2 (11.6-13) °C

pH:

7.9 (7.5-8.5)

Dissolved oxygen:

7.6 (6-8.7) mg/L

Nominal and measured concentrations:

Nominal concentration: 0.03, 0.07, 0.11, 0.18, 0.3 mg/LMeasured (mean) concentration: 0.03, 0.06, 0.11, 0.19, 0.29 mg/L

Details on test conditions:

TEST SYSTEM- Emybro cups (if used, type/material, size, fill volume): 0.2-L glass cylinder, fill volume: ≈ 0.14 L- Test vessel: 10-L glass aquarium - Fill volume: 6.4 L- Volume additions per day: 4.5 L (mean volume addition per day for the complete exposure period)- Type of flow-through: solenoid-activated proportional dilutor systems- No. of fertilized eggs/embryos per vessel: 35 or 40 embryos per cup- No. of vessels per concentration (replicates): 2- No. of vessels per control (replicates): 2- Biomass loading rate: 2.532 g/L (mean loading (g wet weight/L) at the end of the exposure period for control organismsTEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Non-chlorinated deep well located at The Johns Hopkins University Applied Physics Laboratory (JHU/APL) in Shady Side, MD.- Total organic carbon: 19 mg/L- Metals: Sb, As, Be, Pb, se, Tl < 0.005 mg/L, Cd < 0.001 mg/L, Cr < 0.05 mg/L, Cu < 0.02 mg/L, Hg < 0.0002 mg/L, Ni < 0.2 mg/L, Ag <0.01 mg/L, Zn < 0.04 mg/L- Alkalinity: 98 (72-140) mg CaCO3/L- Conductivity: 334 (300-370) µmhos/cm- The water used in was analyzed for 43 Priority Pollutants, eight nonpriority, but "Hazardous" substances, 26 pesticides, and 13 metals. None of them were detected with detection limits of 2, 2, 0.1 µg/, and <0.2 - <0.0002 mg/, respectively.- Intervals of water quality measurement: A comprehensive chemical analysis of the well water was conducted two times during the study. The analyses were separated by approx. 18 months.OTHER TEST CONDITIONS- Photoperiod: 16-h light:8-h dark- Light intensity: fluorescent lights, 60-85 foot candles at the surface of the culture vesselsEFFECT PARAMETERS MEASURED: hatching success (after 8-day exposure), survival of fry (after 60 days of post-hatch exposure), growth of fry (after 60 days of post-hatch exposure)POST-HATCH DETAILS- post-hatch fish were observed daily for developmental abnormalities and mortality- total lenght, blotted wet weight and dry weight were determine for all fish from all treatment

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: dry weight

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: dry weight

Details on results:

- A total of 9 fry had obvious deformities at the end of the 60-d exposure. Seven fry (1 died before the end of the 60-d exposure) had curved spinal cords and two had extremely dark pigmentation. The deformed fry were observed at the three highest test concnetrations.

Reported statistics and error estimates:

Arc-sine square root trasformations were made on survival and hatching success raw data before further data analyses were performed.Both transformed and raw data were then subjected to a chi-square test of normaliy and Bartlett´s test for homogeneity of variance. When the data sets met the assumptions of normality and homogeneity of variance, a parametric statistic was used. Dunnett´s test was used when the number of replicates was constant among treatments. The Kruskal-Wallis procedure was used to compare differences between sample means when less than four replicates per treatment were used.A minimum probability level of 0.05 was used for all tests.

Conclusions:

NG did not affect percent hatch after 8 d of exposure or fry survival after 60 d of post-hatch exposure at concentratioins up to 0.29 mg/L. The total lenght of fry after 60 d of post-hatch exposure was not affected at concentartions up to 0.29 mg/L. Wet weight was significantly reduced at a concentration of 0.11 mg/L, no effect occurred at 0.06mg/L. Dry weight was significantly reduced at a concentration of 0.06 mg/L, no effect occured at 0.03 mg/L. The LOEC and NOEC based on dry weight are 0.06 and 0.03 mg/L, respectively.

Reference 3

Endpoint:

fish life cycle toxicity

Data waiving:

exposure considerations

Justification for data waiving:

other:

Description of key information

Testing is omitted based on Annex XI section 3 of REACH. Exposure of aquatic invertebrates is excluded throughout the whole life cycle of the substance for manufacture and all identified uses. Detailed explanations are attached in section 13 of Iuclid dossier.