Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was published online on 05 Decemebr 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
other: US Environmental Protection Agency. 2002. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, 4th ed. EPA 821/R-02/013. Washington, DC.
Deviations:
not specified
GLP compliance:
not specified
Remarks:
Study conducted to a high scientific standard and findings presented in a comprehensive test report.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Holston Army Ammunition Plant
- Appearance: dry yellow powder
- Purity: 95%
Analytical monitoring:
yes
Details on sampling:
In-water and out-water samples were collected daily and submitted to the US Army Institute of Public Health for determination of concentration by gas chromatography (described in Analytical method). Water-quality parameters were assessed daily on
Inter- and intraspecies sensitivity to dinitroanisole Environ Toxicol Chem 34, 2015 403 both in- and out-water samples.
Laboratory control samples and duplicates and matrix spike and duplicates were analyzed with test samples at a frequency of 5%. In the modified 8330 B method, the relative standard deviation values ranged from 15.3% to 17.9% for DNAN. Therefore, acceptance limits were set to 80% to 120% (typical sample variability is 5–10%).
Vehicle:
yes
Remarks:
Moderately Hard Water
Details on test solutions:
The bioassays consisted of 5 to 6 test nominal concentrations (mg/L) 25, 12.5, 6.3, 3.1, 1.6, and 0.8 DNAN.
Test organisms (species):
other: Ceriodaphnia dubia and Daphnia magna
Details on test organisms:
Ceriodaphnia dubia were obtained from in house cultures (originally purchased from ECTesting) and reared according to guidance [26]. Three different D. pulex populations were obtained from an organism culture laboratory (ECTesting) or recently field-collected sources (H. Webb, St. Louis, MO, USA; A. Beckerman, University of Sheffield, Sheffield, UK). All D. pulex were cultured in-house for at least 11 wk prior to toxicity testing. Hereafter, the 3 D. pulex populations are referred to as population A, population B, and population C.

Genotyping
The 3 D.pulex populations (A, B,and C) were confirmed to be D. pulex using a dichotomous key (http://cfb.unh.edu/cfbkey/ html/anatomy/daphnia/daphnia.html) and through genotyping. Genotyping was determined by retrieving 150 small sequence repeat–containing D. pulex microsatellite sequences from the National Center for Biotechnology nucleotide database (http:// www.ncbi.nlm.nih.gov/) as potential genotyping markers, for which 28 primer sets (fused with a leading M13 sequence) were designed, each targeting a unique microsatellite sequence.
Test type:
static
Water media type:
freshwater
Remarks:
Moderately hard reconstituted water served as the diluent and control
Limit test:
no
Total exposure duration:
6 d
Remarks on exposure duration:
The chronic C. dubia bioassay was a 3-brood test. In this test, all of the control animals had 3 broods after 6d. The, 3 D. pulex chronic tests were conducted for 9d to 11d as this period was required to acquire 3 broods from the3D.pulex populations.
Hardness:
Moderately hard
Test temperature:
25”C
Conductivity:
18.3 MV ohm cm
Nominal and measured concentrations:
The average concentrations measured at test initiation and termination were used.
Details on test conditions:
Chronic toxicity tests using C.dubia (1 test) were conducted in accordance with the US EPA at 25°C. Bioassays consisted of 5 to 6 test nominal concentrations (mg/L) 25, 12.5, 6.3, 3.1, 1.6 and 0.8 DNAN. Both chronic toxicity methods involved daily, static water renewals (100% for C. dubia) using freshly prepared DNAN solution. In-water and out-water samples were collected daily and submitted to the US Army Institute of Public Health for determination of concentration by gas chromatography (described in Analytical method). Water-quality parameters were assessed daily on both in- and out-water samples.

The chronic C. dubia bioassay was a 3-brood test. In this test, all of the control animals had 3 broods after 6d. Each exposure concentration consisted of 10 replicates (20-mL scintillation vials containing 20mL test medium and 1 test animal). A daily feeding ration mixture consisting of half Pseudokirchneriella subcapitata and half yeast, cereal leaves, and trout chow fish food was supplied. Measurement endpoints for the C. dubia test were survival and reproduction. There is no standard chronic method for assessing the toxicity of compounds to D. pulex available from the USEPA. However, chronic toxicity exposures of D. pulex were desirable for a subsequent toxicogenomic analysis method developed specifically to evaluate aquatic exposures to DNAN. Daphnia pulex is an excellent biological model for toxicogenomics because it has greater tissue mass than C. dubia and a better-characterized genome than other cladocerans. Thus, 3 D. pulex chronic tests were conducted in basic accordance with the chronic C. dubia 3-brood test method [27], with the following exceptions.Testswereconductedat258Cfor 9d to 11d as this period was required to acquire 3 broods from the 3 D.pulex populations. In addition,a larger chamber (40mL test medium in 50-mLglass beakers) was used for test replicates (n = 10) as these conditions resulted in better control performance. Feeding rations, water exchanges, and test endpoints were as previously described for the C.dubia test. All 3 obtained populations of D. pulex were used to test the chronic toxicity of DNAN. Additional D. pulex chronic toxicity tests were conducted using population B to compare potential toxicity differences between the sonication and magnetic stirring methods.
Reference substance (positive control):
not specified
Key result
Duration:
6 d
Dose descriptor:
NOEC
Remarks:
Ceriodaphnia dubia
Effect conc.:
> 24.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Survival
Key result
Duration:
6 d
Dose descriptor:
LOEC
Remarks:
Ceriodaphnia dubia
Effect conc.:
> 24.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Survival
Key result
Duration:
6 d
Dose descriptor:
other: MATC
Remarks:
Ceriodaphnia dubia
Effect conc.:
> 24.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Survival
Key result
Duration:
6 d
Dose descriptor:
LC50
Remarks:
Ceropdaphnia dubia
Effect conc.:
> 24.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Survival
Key result
Duration:
6 d
Dose descriptor:
NOEC
Remarks:
Ceropdaphnia dubia
Effect conc.:
6.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Sublethal
Key result
Duration:
6 d
Dose descriptor:
LOEC
Remarks:
Ceropdaphnia dubia
Effect conc.:
12.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Sublethal
Key result
Duration:
11 d
Dose descriptor:
NOEC
Remarks:
Daphnia pulex (mean Pop. A-C)
Effect conc.:
>= 7.2 - <= 14.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Survival
Key result
Duration:
11 d
Dose descriptor:
LOEC
Remarks:
Daphnia pulex (mean Pop. A-C)
Effect conc.:
>= 14.4 - <= 28.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Survival
Key result
Duration:
11 d
Dose descriptor:
other: MATC
Remarks:
Daphnia pulex (mean Pop. A-C)
Effect conc.:
16.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Survival
Key result
Duration:
11 d
Dose descriptor:
LC50
Remarks:
Daphnia pulex (mean Pop. A-C)
Effect conc.:
>= 13.7 - <= 21.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Survival
Details on results:
Genotyping
Genotyping of the 3 D. pulex populations confirmed species and confirmed that the clones collected from each population were genetically distinct.

Chemical stability and fate
Dinitroanisole was stable under static conditions for at least 48h in both the absence and the presence of test organisms. Analysis of the concentration and stability of DNAN in both filtered and unfiltered moderately hard reconstituted water over a 48-h period indicated that the magnetic stirring method fully dissolved the DNAN to a stable concentration. No significant differences in DNAN concentration were observed between preparation methods (unfiltered vs filtered) or over time.

Chronic toxicity
Acceptability criteria for survival (>=80%), growth( >= 0.25mg dry wt in controls; actual mean = 0.421 ± 0.044 mg), and reproduction endpoints (at least 60% of control organisms achieving third brood and an average of 15 or more neonates per surviving adult) and water quality were met for all chronic toxicity bioassays. Because the chronic D. pulex bioassay is not standardized, the acceptability criteria for the C. dubia method were adopted and achieved. Dinitroanisole concentrations were shown to be stable between each of the daily water exchanges; thus, the average concentrations measured for all water exchanges from test initiation to termination were used to generate toxicity reference values. Two chronic D. pulex tests (population B) were conducted to compare the relative toxicity of preparing DNAN by sonication versus magnetic stirring, provided that yellow-colored water was observed after sonication. Lower LC50 values for the survival endpoint were determined for sonicated DNAN (12.5mg/L [9.6–16.4–mg/L]) relative to stirred DNAN (21.6mg/L [18.5–25.1mg/L]). However, IC25 values for the reproduction endpoint for the sonicated (3.0mg/L [2.1– 4.1mg/L]) and stirred (3.1mg/L [1.7–7.7mg/L]) dinitroanisole were very similar and not significantly different, suggesting that sublethal toxicity was not altered by preparation method. Because the sonication method resulted in yellowish water, the stirring method was used for subsequent exposures.

The survival dose–response curves for all 3 test species, including the 3 D. pulex populations, indicated little impact on survival below 10mg/L (Table 2). The C.dubia survival endpoint was least sensitive of the 3 species tested; no LC50 in the chronic test could be generated up to 24.2mg/L.

Effects were observed for D. pulex reproduction (all 3 populations) at lower DNAN concentrations (LOEC range, 1.8–3.6mg/L) relative to the 6-d C. dubia endpoint (LOEC, 12.2mg/L). Lower IC25 values for D. pulex reproduction (1.2– 3.1mg/L) further support that the D. pulex reproduction endpoint was more sensitive to DNAN than C. dubia reproduction (IC25, 8.2mg/L). The D. pulex test (9–11 d) did involve a longer exposure duration than the C. dubia test (6 d). There were no significant differences in sublethal toxicity (IC25) between the 3 D. pulex populations, although 1 population (populationC) produced significantly fewer neonates relative to the other 2 populations. Testing of population B did result in a significantly higher IC50 relative to the other 2 populations. The acute LC50 and chronic IC50 values generated were used to plot a species sensitivity distribution.

Table 2. Summary of toxicity reference values (mg/L) for 2,4-dinitroanisole generated from chronic aquatic toxicity bioassays

     Survival            Sublethal             
 Test species  Source  NOEC  LOEC  MATC  LC50b  NOEC  LOEC  MATC  IC25b  IC50b
 Ceriodaphnia dubia  In-house  >24.2  >24.2  >24.2  >24.2  6.2  12.2  8.7  8.2 (7.4–8.7)  10.6 (10.0–11.2)
 Daphnia pulex  Pop. A  7.2  14.4  10.2  13.7 (7.5–24.9)  1.7  3.6  2.5  2.3 (1.7–2.9)  3.4 (2.9–4.5)
 Daphnia pulex  Pop. B  14.4  28.6  20.3  21.6 (18.5–25.1  1.8  3.6  2.5  3.1 (1.7–7.7)  9.6 (6.2–11.0)
 Daphnia pulex  Pop. C  14.2  28.5  20.1  18.8 (16.1–21.9)  >1.8  1.8  NA  1.2 (0.5–2.9)  2.7 (1.0–3.7)

b = Values in parentheses represent 95% confidence intervals.

NOEC = no-observed-effect concentration;

LOEC = lowest-observed-effect concentration;

MATC = maximum acceptable toxicant concentration;

LC50 = 50% lethal concentration;

IC25/IC50 = 25% and 50% inhibitory concentrations;

Pop. = population;

NA = not applicable.

Validity criteria fulfilled:
yes
Conclusions:
It was concluded that the 11-day toxicity (NOEC) of DNAN ranged from 7.2 to 14.4 mg/L for Daphnia pulex and was 24.2 mg/L for Ceriodaphnia dubia.

Description of key information

The 11-day toxicity (NOEC) of DNAN was found to range from 7.2 to 14.4 mg/L for Daphnia pulex and was 24.2 mg/L for Ceriodaphnia dubia.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
7.2 mg/L

Additional information

A determination of the chronic toxicity to aquatic invertebrates of DNAN was conducted (Kennedy, A.J et al, 2014). Tests were conducted in accordance with standard guidance (U. S. Environmental Protection Agency (USEPA), 2002, EPA-821-R-02-013.

Groups of Ceriodaphnia dubia and Daphnia pulex were exposed to nominal concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8 mg/L of DNAN.

It was concluded that the 11-day toxicity (NOEC) of DNAN ranged from 7.2 to 14.4 mg/L for Daphnia pulex and was 24.2 mg/L for Ceriodaphnia dubia.