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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 Nov 2012 to 16 Oct 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
See Deviations from guideline in "Any other information on materials and methods incl. tables"
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations and controls - taken at initiation, beginning and end of longest renewing cycle each week, and termination
- Sampling method: Collected from mid-depth, and placed in glass vials with 20 µL of sodium azide (1% w/v)
- Sample storage conditions before analysis: Refrigerated
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Prepared every 2 - 3 days individually. Calculated amount of test substance was added to dilution water, and mixed using a magnetic stir bar on a stir plate. Vortex depth was maintained at 10%, and mixed for 49 hrs. The test solution was then allowed to settle for 60 - 90 mins.
- Evidence of undissolved material: No evidence of precipitation. Light green coloration of test substances atributed to alga used for feeding.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: Wildlife International
- Age: < 24 hrs

FEEDING DURING TEST
- Food type: yeast, cereal grass media, trout chow, green algae, vitamin solution
- Amount: 0.5 mL trout chow, 1.0 mL algae, 0.40 mL vitamin solution
- Frequency: Daily

ACCLIMATION
- Acclimation period: 2 weeks
- Health during acclimation (any mortality observed): No signs of disease or ephippia

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Transfers made below water surface using wide-bore pipettes.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
140 - 148 mg/L as CaCO3
Test temperature:
19.9 - 20.9°C
pH:
8.1 - 8.5
Dissolved oxygen:
6.4 - 8.6 mg/L
Nominal and measured concentrations:
Water accommodated fraction: 0.01, 0.1, 1.0, 10.0 mg/L (No test substance was detected in any sample during the analytical monitoring due to the low water solubility of the test substance. Results were therefore reported using the water accommodated fraction).
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Type (delete if not applicable): Covered with plastic petri dishes
- Fill volume: 200 mL
- Renewal rate of test solution (frequency/flow rate): 2 - 3 days
- No. of organisms per vessel: 1
- No. of vessels per concentratio: 10
- No. of vessels per control: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 40 m deep well water, passed through a sand filter and aerated, then filtered through a 0.45 µm filter and sterilized with UV.
- Alkalinity: 176 - 182 mg/L as CaCO3
- Conductivity: 365 - 409 µS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 hrs light/8 hrs dark, with 30 minute transition period
- Light intensity: Similar to natural sunlight

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality, onset of reproduction (3 times/week), signs of toxicity, body length, dry weight

VEHICLE CONTROL PERFORMED: Yes
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
other: NOEL
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
not specified
Duration:
21 d
Dose descriptor:
other: LOEL
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Duration:
21 d
Dose descriptor:
other: MATL
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Remarks on result:
other: not calculable
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: not calculable
Details on results:
An overview of the results is provided in Table 1 in 'Any other information on results incl. tables'

- Mortality of parent animals: Survival of control was 95%, survival in the 0.10 mg/L group was 80%, and 90% in the 10 mg/L group. 100% survival was seen in the 0.01 and 1.0 mg/L groups.
- No. of offspring produced per day per female: Average of 270 offspring per female throughout the study in the negative control group, and an average of 281, 306, 274, and 303 in each respective treatment group.
- Body length and weight of parent animals: 4.8 mm and 1.11 mg average in negative control. 4.6, 4.8, 4.6, and 4.8 mm, and 1.13, 1.19, 1.11, 0.99 mg in respective test groups. Some daphnids in the 10 mg/L treatment group were excluded from dry weight measurements due to the presence of eggs in brood pouches
- Type and number of morphological abnormalities: Paleness in one daphnid in negative control group on Day 3, normal appearance in all other daphnids throughout the study.
- Type and number of behavioural abnormalities: Lethargy in one daphnid in negative control group on Day 3.
- Time to first brood release or time to hatch: Day 7, 8, or 9
- Other biological observations: One immobile neonate observed in the negative control group and in the 1.0 mg/L group on Day 12.
Reported statistics and error estimates:
No statistical decrease in exposure groups as compared to negative controls (p > 0.05). No statistically significant decrease in mean neonate production in any test group (p > 0.05). No statistically significant decrease in dry weight or body length (p > 0.05).

Table 1. An overview of the results.

Concentration

(mg/L WAF)

Neonate Production

(Treatment Mean ± Std. Dev.)

Length

(Mean ± Std. Dev.)

(mm)

Dry Weight
(Mean
± Std. Dev.)

(mg)

Negative control

270 ± 23

4.8 ± 0.13

1.11 ± 0.147

0.01

281 ± 31

4.6 ± 0.20

1.13 ± 0.0917

0.10

306 ± 20

4.8 ± 0.21

1.19 ± 0.0958

1.0

274 ± 22

4.6 ± 0.095

1.11 ± 0.131

10

303 ± 21

4.8 ± 0.12

0.99 ± 0.184

Validity criteria fulfilled:
yes
Conclusions:
Based on the findings, the 21-day NOEL of Daphnia magna exposed to Benezene 10-13, alkyl derivatives was determined to be ≥ 10 mg/L (WAF), and the EL50 was determined to be > 10 mg/L (WAF) for both immobilization and reproduction.
Executive summary:

This study determined the long-term toxicity of Benzene 10 -13, alkyl derivatives to the freshwater aquatic invertebrates, Daphnia magna. This study was conducted according to OECD TG 211 and compliance with GLP. The daphnia were exposed to the test substance in a semi-static system and at concentrations of 0.01, 0.1, 1.0, 10.0 mg/L for 21 days. Both immobilization and reproductive parameters were monitored. Due to the low solubility of the test substance, no test substance was detected in the analytical monitoring. Thus, the results are therefore reported as a water accommodated fraction (WAF). After 21 days exposure, no statistically significant (p > 0.05) effect were observed on the mortality, neonate production, dry weight or body length of the tested daphnia.

Based on the findings, the 21-day EL50 was determined to be > 10 mg/L (WAF) for both immobilization and reproduction, the LOEL was determined to be > 10 mg/L (WAF) and the 21-day NOEL was determined to be ≥ 10 mg/L (WAF).

Description of key information

All available data was assessed. The lowest effect level (NOEC = 0.0075 mg/L) was found in the 21-day chronic study on Daphnia magna (Gledhill et al.,1991). However, in this study the solubility of LAB was artificially enhanced by using acetone as a solvent. In accordance with the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, ENV/JM/MONO (2000)6 and ECHA Guidance on information requirements and chemical safety assessment (version 4.0, June 2017), Chapter R7b, Table R.7.8-3, aquatic toxicity testing of a UVCB substance which is only partially soluble in water should preferably be conducted using a water-accommodated fraction (WAF) of the UVCB and solvents should be avoided to prevent unrealistic conclusions. In addition, the the solvent concentration (1 mg/L) in this study was above the recommended level for solvent addition (< 0.1 mL/L) (OECD Guideline No. 211). Based on these reasons, the NOEC value from this study was considered not reliable for the derivation of PNECs and probably overestimake the actual effect.

In comparsion, the 21-day long-term toxicity test on freshwater invertebrate (Daphnia magna) used the water accommodated fraction method according to OECD guideline No.211. It was considered to be the key study and selected for CSA. The other studies were included as supporting information. The NOEL was determined to be 10 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
10 mg/L

Additional information

The 21-day study examined the effects of chronic exposure of Daphnia magna to the test substance by using the water accommodated fraction (WAF) method (OECD Guideline 211). Ten first-instar Daphnia (< 24 h old) were individually placed in test chambers with test solution (200 mL) containing 0, 0.01, 0.1, 1.0 or 10.0 mg/L (WAF) of the test substance. Test solutions were changed every 2-3 days. Effect parameters measured included mortality, onset of reproduction (measured three times per week) signs of toxicity, body length and dry weight. Due to the low water solubility of the test substance, no test substance was detected in the analytical monitoring (SPME-GC-FID). The results are therefore reported as a WAF (nominal concentration). The 21-day EL50 for Daphnia magna exposed to LAB was >10 mg/L (WAF) for both immobilization and reproduction. The 21-day NOEL for Daphnia magna was 10 mg/L (WAF) and the 21-day LOEL was >10 mg/L (WAF). Based on these results LAB is not classified for chronic aquatic toxicity.

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