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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
5 to 7 August 2009
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
Draft version
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dilution series from stock solution provided by sponsor
- Eluate: water
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): none
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
- Other relevant information:
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Springborn Smithers laboratory culture
- Justification for species other than prescribed by test guideline: n.a.
- Age at study initiation (mean and range, SD): <24h
- Weight at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD):not reported
- Stage and instar at study initiation: not specified
- Valve height at study initiation, for shell deposition study (mean and range, SD): not reported
- Peripheral shell growth removed prior to test initiation:not reported
- Method of breeding: continuos culture
- Source: house breed
- Age of parental stock (mean and range, SD): no
- Feeding during test: no
- Food type:
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period: no
- Acclimation conditions (same as test or not): same
- Type and amount of food: Ankistrodesmus falcatus, 4 x 107 cells/mL) at a rate of 2.0 mL, and 0.50 mL of a combination of yeast, cereal leaves and flaked fish food suspension (YCT) per vessel daily.

QUARANTINE (wild caught) NOT APPLICABLE
- Duration:
- Health/mortality:

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
no
Hardness:
Total hardness as CaCO3: 38 to 50 mg/L
Test temperature:
19 to 22 ºC,
pH:
pH: 6.7 to 6.9
Dissolved oxygen:
7.5 to 9.6 mg/L,
Salinity:
Total alkalinity as CaCO3: 19 to 22 mg/L
Conductivity:
350 to 370 μmhos/cm
Nominal and measured concentrations:
nominal:
0.63, 1.3, 2.5, 5.0 and 10 mg a.i./L

measured (geom mean)
0.47, 1.4, 2.4, 4.9 and 9.5 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: square glass battery jars
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 1600 mL
- Volume of solution: 1400 mL per vessel
- Aeration: not specified
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): 90% test solution replacement rate of approximately nine hours
- No. of organisms per vessel: 10/ conc
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness
- Light intensity: 40 to 76 footcandles (430 to 820 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility
VEHICLE CONTROL PERFORMED: no
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 9.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
ca. 2.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no
- Observations on body length and weight: not measured
- Other biological observations: not reported
- Mortality of control: not reported
- Other adverse effects control:not reported
- Immobilisation of control: not reported
- Abnormal responses: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: no, but pH shifting by the test substance did allow higher test concentrations.
Results with reference substance (positive control):
not reported

Since no concentration tested resulted in ≥ 50% immobilization, the 48-hour EC50 value was empirically estimated to be > 9.5 mg a.i./L, the highest geometric mean measured concentration tested. The No-Observed-Effect Concentration (NOEC) was determined to be 2.4 mg a.i./L.

Further testing to define an EC50 value was not performed since the highest nominal concentration tested (i.e., 10 mg a.i./L) represents the highest test concentration at which the pH of the exposure solutions was appropriate for the survival of Daphnia magna.

Validity criteria fulfilled:
yes
Conclusions:
The study principally fulfills the quality standards, but test results obtained during the test did not let conclude on a definite EC50, as the highest tested concentration was found to be below the point from where the EC50 could be derived. The observed no effect concentration however could be assessed to be 4.9 mg a.i./L.
Executive summary:

During the test period no immobilisation in the tested concentrations 0.47, 1.4 and 2.4 mg a.i./L was observed. Observed effects occured in the 4.9 mg/L concentration and in the 9.5 mg/L concentration. The latter was determined to be the NOEC whereas the 9.5 mg/L concentration did not reach a nkuber from what the EC50 value could be derived.

Further testing to define an EC50 value was not performed since the highest nominal concentration tested (i.e., 10 mg a.i./L) represents the highest test concentration at which the pH of the exposure solutions was appropriate for the survival of Daphnia magna.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed to a reliable method but not to GLP
Qualifier:
no guideline followed
Principles of method if other than guideline:
Acute 24 and 48 h Ceriodaphnia range-finding studies were conducted using the test substance stock solutions that were either pH-adjusted or non-adjusted to discern the toxicity that could be attributed to the acidity of the test substance, and to select between the pH treatment alternatives for exposure media in the definitive test.
GLP compliance:
not specified
Remarks:
Although GLP status is not specified, this study is very detailed and was performed by the Environmental Toxicology Branch, U.S. Army Edgewood Chemical Biological Center for the U. S. Army Center for Health Promotion and Preventive Medicine.
Specific details on test material used for the study:
- Source BAE Systems
- Lot No. BAE07B305-001
- Purity: 99.6%
Analytical monitoring:
yes
Details on sampling:
None stated
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetonitrile with 0.1% trifluoroacetic acid

No additional data
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Strain: Ceriodaphnia dubia

No additional data
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None stated
Hardness:
None stated
Test temperature:
25℃
pH:
pH-adjusted media pH: 7.5
pH non-adjusted media pH: 3.1-7.5
Dissolved oxygen:
None stated
Salinity:
None stated
Nominal and measured concentrations:
Measured concentrations: 0 (control), 62, 124, 246, 479, and 881 mg/L for the pH-adjusted treatment groups; 0 (control), 33, 64, 127, 248, 489, and 904 mg/L for the non-pH-adjusted treatment groups.
Details on test conditions:
None stated
Reference substance (positive control):
not specified
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
830 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 807-854 mg/L, pH-adjusted media
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
460 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: pH-adjusted media
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
66 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: non-pH-adjusted media
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
62 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: non-pH-adjusted media
Details on results:
The results showed that adjusting the pH of the test substance treatment groups decreased toxicity by 92% in the 24 h exposure test, and by 87% in the 48 h exposure test
Results with reference substance (positive control):
No information provided
Reported statistics and error estimates:
No information provided
Validity criteria fulfilled:
yes
Conclusions:
Increasing the pH of Ceriodaphnia media containing the test substance (to pH 7.5; the pH of Ceriodaphnia media without test substance) decreased the toxicity in acute Ceriodaphnia tests. The LC50 values differed by approximately an order of magnitude between the two pH treatments of the exposure media containing the test substance (pH unadjusted, or pH adjusted to accommodate the respective test substance content).
The 48h LC50 to Ceriodaphnia dubia is 460 mg/L for pH-adjusted media and 62 mg/L for non-pH-adjusted media.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The result is calculated data as modeled by EPIWIN.
Justification for type of information:
QSAR prediction: migrated from IUCLID 5.6
Principles of method if other than guideline:
Model for determining aquatic toxicity based on Log Kow and water solubility.
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
222.878 mg/L
Conc. based on:
test mat.
Remarks on result:
other: This value is predicted for Phenols class.
Conclusions:
Based on the prediction results described, the test substance, NTO, was predicted to have LC50 of 222.878 mg/L after 48 hours exposure.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
other: Published paper publically available
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
other: US Environmental Protection Agency. 2002. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms,
Version / remarks:
5th ed. EPA 821/R-02/012. Office of Water, Washington
Deviations:
not specified
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Holston Army Ammunition Plant (BAE Systems, Kingsport, TN, USA. Btach no not stated
- Expiration date of the lot/batch: Not stated
- Purity test date: >95%

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Preliminary purification step (if any): none
- Final preparation of a solid: NTO was dissolved directly into a standard synthetic freshwater bioassay medium (moderately hard reconstituted water [MHRW]]) by
magnetic stirring overnight. The pH of the resultant solution was pH2.9, as the test requires pH 6.5 - 9.0 the pH of the NTO solution was increased (to 7.7–8.2) using reagent grade NaOH. Test solutions and pHmodifications, where applicable, were tested within 24 h.

FORM AS APPLIED IN THE TEST - see above

Analytical monitoring:
yes
Details on sampling:
Test solutions and pHmodifications, where applicable, were tested within 24 h. All test solutions were measure at 0 and 48 h during the test.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
NTO was dissolved directly into a standard synthetic freshwater bioassay medium (moderately hard reconstituted water [MHRW]]) by magnetic stirring overnight. The pH of the resultant solution was pH2.9, as the test requires pH 6.5 - 9.0 the pH of the NTO solution was increased (to 7.7–8.2) using reagent grade NaOH. Test solutions and pHmodifications, where applicable, were tested within 24 h.

- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): stock solution of 1500 mg/l prepared then for the bioassay experiment a furthe 6 concentrations (in order to reach a concentration no-effect level) were created from a 50% serial dilution (100%, 50%, 25%, 12.5%, 6.25%, etc.), using MHRW as the diluent and control.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not stated
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Test organisms were obtained from in-house cultures, originally purchased from a commercial source (ECTesting). Ceriodaphnia dubia was selected because of its relatively high sensitivity to aquatic contaminants and its routine use in National Pollutant Discharge
Phototoxicity of insensitive munitions Elimination System permit whole effluent toxicity testing.
- Age at study initiation (mean and range, SD): Not stated
- Weight at study initiation (mean and range, SD): Not stated
- Length at study initiation (length definition, mean, range and SD): Not stated
- Stage and instar at study initiation: Not stated
- Source: see above
- Age of parental stock (mean and range, SD): Not stated
- Feeding during test Not stated
- Food type: Not stated

ACCLIMATION
- Acclimation period: Not stated
- Acclimation conditions (same as test or not): Not stated
- Type and amount of food: Not stated
- Feeding frequency: Not stated
- Health during acclimation (any mortality observed): Not stated

Test type:
static
Water media type:
other: standard synthetic freshwater bioassay medium (moderately hard reconstituted water [MHRW]
Limit test:
no
Total exposure duration:
48 h
Hardness:
80-100 mg CaCO3/L
Test temperature:
25 ± 1 C

pH:
7.9 - 8.3
Dissolved oxygen:
Not stated
Nominal and measured concentrations:
Nominal concentrations for the non photo-degradation test were 0, 24, 47, 96, 192, 384, 768 and 1536 mg/L and measured concentrations (at 0h) were 20.8, 41.0, 90.3, 178.8, 365.7, 746.5 and 1507.6 mg/L
Nominal concentrations for the first photo-degradation test were 3, 6, 12, 24, 47, 94, 188, 377, 754 and 1507 mg/L and measured concentrations (at 0h) were 2.6,5.1, 10.2, 20.3, 40.8, 85.5, 173.6, 345.4, 726.7 and 1451.7 mg/L
Nominal concentrations for the second photo-degradation test were 0, 0, 0, 0, 0, 0, 0, 2, 14, 55 and 110 mg/L and measured concentrations (at 0h) were 0, 0.2, 0.3, 0.6, 1.3, 2.6, 5.1, 10.2, 19.7, 41.9 and 91.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 20mL glass scintillation vial
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:
- Volume of solution: Not stated
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate):n/a
- No. of organisms per vessel:5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates):
- Biomass loading rate: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: moderately hard reconstituted water [MHRW] prepared for study use
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity: 57-64 (nominal)
- Ca/mg ratio: 1:1 (nominal)
- Conductivity:
- Salinity:
- Intervals of water quality measurement: 0 and 48h

OTHER TEST CONDITIONS
- Adjustment of pH: pH adjusted to 7.2 - 7.8
- Photoperiod: 16 h light, 8 h darkness
- Light intensity: normal laboratory light


VEHICLE CONTROL PERFORMED: yes

Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
799.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
non-photodegraded test
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 111 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
photo degraded test 2
Details on results:
- Behavioural abnormalities:
- Observations on body length and weight:
- Other biological observations:
- Mortality of control:
- Other adverse effects control:
- Immobilisation of control:
- Abnormal responses:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:
Reported statistics and error estimates:
not reported

The study reveals a negligible toxicity of the parent compound, and a far higher toxicity of photodegradation products. However, these have not been chemically quantified sufficiently and consequently not been reported.

Validity criteria fulfilled:
yes
Conclusions:
The presented paper deals with the abiotic (photo) degradation and biotic (aquatic) toxicity of these degradation products of various explosives. The results are qualtitative pointing to a higher toxicity of degradation products then of the parental substance NTO. But, as chemical analysis for these degradation products is missing, just the parental substance can be assessed.

Description of key information

According to the computer program EPISUITE, the test substance, NTO, was predicted to have LC50 of 222.878 mg/L after 48 hours exposure.

As stated in the published data (Haley Mark V., Kuperman Roman G., Checkai Ronald T. ) the 48h LC50 to Ceriodaphnia dubia is 460 mg/L for pH-adjusted media and 62 mg/L for non-pH-adjusted media.

Kennedy et al .reported for NTO a relatively low toxicity of around 800 mg/L and a so-called "NSDS" ( not significantly different from the control) comparable to a NOEC of 366 mg/L. In contrast, a BAE study revealed a far lower NOEC of 2.4 mg/L in a very sensible system, what did not let conclude on a EC50 value, as the pH was shifiting towards levels which limited the applicability of the test system for the substance. Accordingly the Kennedy study was assessed as the more relevant in this case.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
62 mg/L

Additional information

The short term toxicity to aquatic invertebrates was determined using the computer program EPISUITE, based onLog Kow and water solubility. The test substance, NTO, was predicted to have LC50 of 222.878 mg/L after 48 hours exposure.

Determination of short term toxicity to aquatic invertebrates, Ceriodaphnia dubia, has been also described in published data (Haley Mark V., Kuperman Roman G., Checkai Ronald T. ) describing the test performed at RESEARCH AND TECHNOLOGY DIRECTORATE. The 48h LC50 to Ceriodaphnia dubia is 460 mg/L for pH-adjusted media and 62 mg/L for non-pH-adjusted media.

From a BAE system study no EC50 could be derived due to to test material inherent pH changing properties.