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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
information not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Information not available.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Guideline:
other: not specified
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: test material obtained from Sigma-Aldrich (St Louis, MO, USA)
Analytical monitoring:
not specified
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a stock solution was prepared in reconstituted water (ISO 1982)
- Controls: reconstituted ISO water
Test organisms (species):
other aquatic worm: Freshwater planarian D. japonica
Details on test organisms:
TEST ORGANISM
- Common name: Dugesia japonica
- Justification for species other than prescribed by test guideline: Freshwater free-living planarians are common aquatic invertebrates in both unpolluted rivers and lakes, and play an important role in aquatic ecosystem by acting as carnivores, omnivores, or detritivores. Freshwater planarians have been suggested as suitable
test organisms for various types of short-term toxicity bioassays. In particular, they are sensitive to different types of environmental pollutants and can be easily maintained in laboratory inexpensively. For example, freshwater planarians, Dugesia japonica, showed a similar sensitivity to acute toxicity of different surfactants compared with the most common invertebrate test species, Daphnia magna.
- Source: D. japonica, were collected from Nan-shi stream located in Wu-Lai (24 790 N, 121 520 E) of northern Taiwan. Since then, the animals have been maintained asexually in dechlorinated tap water at room temperature and were fed with raw chicken liver once a week.
- Feeding during test: No. The test animals were not fed at least 4 days before the start of the toxicity tests and during the whole test duration.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The planarians used for the test had a body length of 0.7 ± 0.1 cm.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
25 ± 1 °C
Nominal and measured concentrations:
0.5 - 50 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 50mL of test solution in a glass beaker :
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (ISO 1982), including 2.5 mmol/L CaCl2, 0.4 mmol/L MgSO4, 0.8 mmol/L NaHCO3, and 77 mmol/L KCl.
- Culture medium different from test medium: Yes, dechlorinated tap water.

OTHER TEST CONDITIONS
- Photoperiod: 12 : 12 h dark/light cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The animals were inspected every 24 h for mortality during the entire 96 h experimental period. The organisms without detectable movement were considered dead and removed from test solution.

VEHICLE CONTROL PERFORMED: no
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits: 1.7 - 2.7 mg/L
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits: 1.7 - 2.7 mg/L
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits: 10.9 - 17.9 mg/L
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Reported statistics and error estimates:
The concentrations that were lethal to 50% of the organisms (LC50) at 24, 48, 72, or 96 h were calculated using trimmed Spearman–Karber analysis with trimmed
Spearman–Karber program (version 1.5) which were obtained from Environmental Monitoring Systems Laboratory (USEPA, Cincinnati, OH). The Pearson’s correlation coefficients were calculated to determine the relationship between acute toxicity of D. japonica and D. magna.
Validity criteria fulfilled:
not specified
Conclusions:
In a study with the aquatic invertebrate Dugesia japonica under static conditions, toxic effects of 2,2-Dihydroxy-4,4-dimethoxybenzophenone (Benzophenone-6) were investigated. The following results were determined for the compound: LC50 (24 h) = 35 mg/L, LC50 (48 h) = 14 mg/L, LC50 (72 h) = 2.1 mg/L, LC50 (96h) = 2.1 mg/L. The LC50 (48h) was regarded as key value, since the test duration of 48 h is recommended for acute toxicity testing on aquatic invertebrates according to relevant standard test guidelines.
Based on this result, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, taking into account the facts that the test item is soluble in water, i.e. above the determined EC50 value, and not readily biodegradable, the test item should be classified as aquatic chronic Cat. 3.
Executive summary:

Within this study fourteen benzophenone-type UV filters and four paraben preservatives were selected to examine their acute toxicities on Dugesia japonica. The 48-h LC50 values for planarians exposed to benzophenone-type UV filters can be ranked as oxybenzone >mexenone >5-chloro-2-hydroxybenzophenone >2,4- dihydroxybenzophenone >hydroxybenzophenone >dioxybenzone>benzophenone >2,2,4,4-Tetrahydroxybenzophenone >4-hydroxybenzophenone >3-hydroxybenzophenone >4,4-dihydroxybenzophenone >2,2-dihydroxy-4,4-Dimethoxybenzophenone >2,3,4-trihydroxybenzophenone >sulisobenzone with a range from 0.9 to 145 mg/L with a similar sequence for the 96 h LC50 values, ranging from 0.5 to 77mg/L. Among all the tested chemicals, oxybenzone was the most toxic and sulisobenzone the least toxic chemical to planarian at each exposure period. Most benzophenone-type UV filters are toxic to aquatic animals with 48 h LC50 values less than 10 mg/L, except for 2,2-dihydroxy-4,4-dimethoxybenzophenone, 2,3,4-trihydroxybenzophenone, and sulisobenzone.

The LC50 (48h) for Dugesia japonica exposed to 2,2-Dihydroxy-4,4-dimethoxybenzophenone (Benzophenone-6, CAS 131-54-4) was determined to be 14 mg/L.

Data source

Reference
Reference Type:
publication
Title:
Acute Toxicity and Ecological Risk Assessment of Benzophenone and N,N-Diethyl-3 Methylbenzamide in Personal Care Products
Author:
Sun H-Q
Year:
2016
Bibliographic source:
International Journal of Environmental Research and Public Health - Open Access Journal (www.mdpi.com/journal/ijerph)

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Chinese National Standards: GB/T 21805-2008 (algae)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Qualifier:
equivalent or similar to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzophenone
EC Number:
204-337-6
EC Name:
Benzophenone
Cas Number:
119-61-9
Molecular formula:
C13H10O
IUPAC Name:
benzophenone
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China)

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
yes
Remarks:
dimethyl sulfoxide (cosolvent)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: BG 11 liquid medium and a Benzophenone stock solution were used to prepare a serial concentration of solution (100 mL).

Test organisms

Test organisms (species):
Chlorella vulgaris
Details on test organisms:
TEST ORGANISM
- Common name: Chlorella vulgaris
- Strain: FACHB-8
- Source (laboratory, culture collection): Freshwater Algae Culture Collection, at the Institute
of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The algae were cultured for three generations in the laboratory before the toxicity experiments.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h

Test conditions

Test temperature:
25 ± 2 °C,
pH:
pH 7.1
Nominal and measured concentrations:
The chemical concentrations of the definitive test were set up based on the results of the pre-test, so the concentration ranges are narrow and have good linear relation to the inhibition rates. Five concentrations in the concentration range of 2.00 - 10.00 mg/L were tested.
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250 mL glass flasks with 100 mL of test solution
- Initial cells density: 1E6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3 (cosolvent)

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
BG 11 medium:
A) Stock solutions for BG-11:
Stock 1:
Na2MG EDTA: 0.1g/liter
Ferric ammonium citrate: 0.6g/liter
Citric acid . 1H2O: 0.6g/liter
CaCl2 . 2H2O: 3.6g/liter
Filter sterilize into a sterile bottle or autoclave

Stock 2:
MgSO4 . 7H2O: 7.5g/liter
Filter sterilize into a sterile bottle or autoclave

Stock 3:
K2HPO4 . 3H2O: 4.0g/liter
or K2HPO4: 3.05g/liter
Filter sterilize into a sterile bottle or autoclave

Stock 5 (Microelements):
H3BO3: 2.86g/liter
MnCl2 . 4H2O: 1.81g/liter
ZnSO4 . 7H2O: 0.222g/liter
CuSO4 . 5H2O: 0.079g/liter
COCl2 . 6H2O: 0.050g/liter
NaMoO4 . 2H2O: 0.391g/liter
or MoO4 (85%): 0.018g/liter

B) For basic BG11 medium combine the following stock solutions:
Stock Solution Per Liter of medium
Stock 1
10 ml
Stock 2
10 ml
Stock 3
10 ml
Na2CO3
0.02g
Stock 5
1.0 ml
NaNO3
1.5g

Combine stocks and adjust pH to 7.5 (use 1.0N HCl). Aliquot into flasks (50 ml/125 ml flask) with cotton stoppers on top and autoclave. After autoclaving and cooling the pH is about 7.1. For solid media add 1% noble agar. For BG-11o don't add NaNO3 .

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The growth medium BG 11 and Benzophenone stock solutions were used to prepare a serial concentration of solutions of Benzophenone (100 mL).
- Culture medium different from test medium: not specified

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Photoperiod: 12 h:12 h light–dark cycle
- Light intensity and quality: 2000–3000 lx,

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : During the period of the experiment, the absorbance of C. vulgaris was measured every 24 h, then it
was converted to cell density to calculate growth rates.
- Determination of cell concentrations: Before the experiment, microscopic counting and spectrophotometry were employed to obtain the linear correlation between the C. vulgaris cells density and absorbance at 680-nm wavelength. This correlation was used to determine the cell density for every 24 h, by the measurement of absorbance (see above). The converted cell denity was used to calculate growth rates.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not specified
- Range finding study: A pre-test was conducted but no details were specified in the publication.
- Test concentrations: 2.00 - 10.00 mg/L
- Results used to determine the conditions for the definitive study: The chemical concentrations were set up based on the results of a pre-test, so the concentration range is narrow and has good linear relation to the inhibition rates.
Reference substance (positive control):
not specified

Results and discussion

Effect concentrations
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
6.86 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 6.40–7.34 mg/L
Reported statistics and error estimates:
IBM SPSS version 20 software (IBM, Armonk, NY, USA) was applied to calculate the 96-h half-maximal effective concentration (EC50) of C. vulgaris

Any other information on results incl. tables

Results

There was no significant difference between the dimethyl sulfoxide control group at the highest concentration and the blank control group for C. vulgaris growth rate by analysis of variance. This indicated that dimethyl sulfoxide had no repression effect on C. vulgaris in this study. All the results were analyzed by comparing the testing to the blank control groups.

 

Acute Toxicity of Benzophenone to Chlorella vulgaris (Growth Inhibition)

The inhibition effect on C. vulgaris growth increased with the increasing in the concentrations.

During the exposure time, although the algal growth rate was inhibited, the algal densities still showed an increasing trend. When the Benzophenone concentration was 10.00 mg/L, there was almost no growth between 48 and 72 h, but rapid growth from 72 to 96 h.

The linear regression equation between Benzophenone concentration and its inhibition ratio was

y = -10.216 + 8.979x, (x : 2.00mg/L - 10.00mg/L)

where x is the Benzophenone concentration, and y is the inhibition ratio on C. vulgaris growth (%).

The 96-h EC50 was calculated as 6.86 mg/L (95% confidence interval: 6.40–7.34 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
The study was performed according to Chinese National Standards (GB/T 21805-2008 (algae)) on Benzophenone without obvious deviations which may have an impact on the validity of the study. Thus, the results were obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable.
Under the conditions of the test, there was a significant growth inhibition of Chlorella vulgaris at concentrations >2.00 mg/L. The following EC-value was determined accordingly after 96 hours: EC50 = 6.86 mg/L.
Executive summary:

In the present acute toxicity sudy according to Chinese National Standards, the toxic effect of Benzophenone and N,N-diethyl-3-methylbenzamide on Chlorella vulgaris,Daphnia magna, and Brachydanio rerio was tested and the ecotoxicological risks were evaluated.

For Benzophenone the 96-h half-maximal effective concentration (EC50) on Chlorella vulgaris was 6.86 mg/L.

.