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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2019 - 24 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006, Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reference substance 001
Cas Number:
67-47-0
Molecular formula:
C6H6O3
impurity 1
Chemical structure
Reference substance name:
Fructose
EC Number:
200-333-3
EC Name:
Fructose
Cas Number:
57-48-7
Molecular formula:
C6H12O6
IUPAC Name:
D-fructose
impurity 2
Chemical structure
Reference substance name:
1-(furan-2-yl)-2-hydroxyethanone
Cas Number:
17678-19-2
Molecular formula:
C6H6O3
IUPAC Name:
1-(furan-2-yl)-2-hydroxyethanone
impurity 3
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
impurity 4
Chemical structure
Reference substance name:
Sodium formate
EC Number:
205-488-0
EC Name:
Sodium formate
Cas Number:
141-53-7
Molecular formula:
CH2O2.Na
IUPAC Name:
sodium formate
impurity 5
Chemical structure
Reference substance name:
Sodium nitrate
EC Number:
231-554-3
EC Name:
Sodium nitrate
Cas Number:
7631-99-4
Molecular formula:
HNO3.Na
IUPAC Name:
sodium nitrate
impurity 6
Chemical structure
Reference substance name:
Glucose
EC Number:
200-075-1
EC Name:
Glucose
Cas Number:
50-99-7
Molecular formula:
C6H12O6
IUPAC Name:
D-glucose
impurity 7
Chemical structure
Reference substance name:
Sodium acetate
EC Number:
204-823-8
EC Name:
Sodium acetate
Cas Number:
127-09-3
Molecular formula:
C2H4O2.Na
IUPAC Name:
sodium acetate
impurity 8
Chemical structure
Reference substance name:
2-furaldehyde
EC Number:
202-627-7
EC Name:
2-furaldehyde
Cas Number:
98-01-1
Molecular formula:
C5H4O2
IUPAC Name:
2-furaldehyde
impurity 9
Chemical structure
Reference substance name:
Sodium 4-oxovalerate
EC Number:
243-378-4
EC Name:
Sodium 4-oxovalerate
Cas Number:
19856-23-6
Molecular formula:
C5H8O3.Na
IUPAC Name:
sodium 4-oxopentanoate
impurity 10
Chemical structure
Reference substance name:
Formaldehyde
EC Number:
200-001-8
EC Name:
Formaldehyde
Cas Number:
50-00-0
Molecular formula:
CH2O
IUPAC Name:
formaldehyde
Test material form:
solid: crystalline
Details on test material:
freeze-dried material
Batch: 1808A-RO-C-I1-FD-Chg#3
Appearance Crystal / brown
Storage: Refrigerator (2 - 8 °C)
Expiring date: 31 December 2019

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test water, test item and algae (three replicates per test concentration)
- Blank control (Bn); containing test water and algae (six replicates)
- Since the test item is soluble, the test solution was prepared by respective dilutions of a stock solution. A stock solution of 117 mg/l test item was prepared by adding the test item to test water in sterile glassware and stirred until dissolution. The respective dilutions of this stock solution were then prepared with test water and sterile glassware. 100 ml of each test media were added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
Pure test water, handled as the test item solutions inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls. For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
- Evidence of undissolved material: Not relevant. Since the test item is soluble, the test solution was prepared by directly dissolving the test item in sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2)

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Test organism: Axenic slope culture of Raphidocelis subcapitata 61.81SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from an axenic slope culture
- Illumination: Continuous (4440–8880 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- Control of sensitivity: twice per year, with potassium dichromate

Study design

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

Test conditions

Hardness:
not reported
Test temperature:
Min: 22.0; Max: 23.0; Mean: 22.5
pH:
7.8 - 9.0
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
The concentrations of 5-(Hydroxymethyl)furfural in the test media were measured by HPLC analysis with UV detection at the beginning and after 24, 48 h and 72 h of exposure. These analyses showed that the test concentrations were correctly dosed. However, the test concentrations decreased over the 72 hours test period and did not remain within 80–120% of the nominal value but ranged from 10% to 73% in the three lowest concentrations and within 80–120%
of the nominal value in the two highest concentrations.
Therefore, the effective concentrations ECx were assessed based on the geometric mean of the measured concentrations of the test item over the entire 72 hours test period, which were 122, 59.7, 27.1, 10.6 and 3.76 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm), capped with air permeable stoppers
- Test medium: Recommended OECD medium
- Preculture: Exponentially growing liquid culture of Raphidocelis subcapitata kept under the same environmental conditions as in the test
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity: the light intensity amounts to about 5000 lux which corresponds to about 40 µE•m-2•s-2
- Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of 5-(Hydroxymethyl)furfural was performed. The results indicated that the test item is well soluble and that the test solutions can be prepared by respective dilutions of a stock solution. The HPLC determinations indicate that the test item was correctly dosed, but the concentrations decreased over the duration of the test. Based on the results of this non-GLP range finding test the following concentrations were selected for the definitive test: 117, 58.5, 29.3, 14.6 and 7.32 mg/l of test item (which correspond to 100, 50.0,
25.0, 12.5 and 6.25 mg/l of the main component 5-(Hydroxymethyl)furfural).

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.
Reference substance (positive control):
not required
Remarks:
Control of sensitivity: None – the culture is re-purchased annually

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
132 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated using a linear regression
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
39.2 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated using a linear regression
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
59.7 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated using a linear regression
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
27.1 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated using a linear regression
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
45.5 mg/L
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: calculated using a linear regression
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.2 mg/L
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: calculated using a linear regression
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
27.1 mg/L
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: calculated using a linear regression
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10.6 mg/L
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: calculated using a linear regression
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: yes (growth rate and yield)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

With respect to the endpoint average specific growth rate, the following effects were observed compared to the control: 47% at 122 mg/l, 19% at 59.7 mg/l, 5% 27.1 mg/l, 1% at 10.6 mg/l and 2% at 3.76 mg/l.
The 72-hour EC50 based on average specific growth rate of 5-(Hydroxymethyl)furfural to Raphidocelis subcapitata was calculated to be 132 mg/l (95% confidence interval: 128–136 mg/l).
The EC10 was calculated to be 39.2 mg/l (95% confidence interval: 37.0–41.3 mg/l). These ECx were computed using a linear regression with a logit link function and a maximum likelihood algorithm (slope = 4.18, Intercept = –8.85, degrees of freedom = 13, p(Chi²) = 1.00, r2 =0.991, p(F) <0.001).
Even though 5-(Hydroxymethyl)furfural induced statistically significant inhibitions of 5%, 1% and 2% at the concentrations of 27.1, 10.6 and 3.76 mg/l, respectively (based on a Williams’ multiple sequential t-test procedure, one-sided smaller, ¿ = 0.05), these effects were not considered as biologically relevant toxic effects, since they are below the allowed coefficient of variation of the average specific growth rates of the controls (7%) as described in the validity criteria.
Therefore, the LOEC with respect to average specific growth rate is 59.7 mg/l and the NOEC is 27.1 mg/l.

With respect to the endpoint yield, the following effects were observed compared to the control: 92% at 122 mg/l, 63% at 59.7 mg/l, 24% at 27.1 mg/l, 7% at 10.6 mg/l and 8% at 3.76 mg/l.
The 72-hour EC50 based on yield of 5-(Hydroxymethyl)furfural to Raphidocelis subcapitata was calculated to be 45.5 mg/l (95% confidence interval: 41.6–49.6 mg/l). The EC10 was calculated to be 16.2 mg/l (95% confidence interval: 12.8–19.3 mg/l). These ECx were computed using a linear regression with a logit link function and a maximum likelihood algorithm (slope = 4.91, Intercept = – 8.15, degrees of freedom = 13, p(Chi²) = 1.00, r2 = 0.926, p(F) <0.001). Even though 5-(Hydroxymethyl)furfural induced statistically significant inhibitions of 7% and 8% at the concentrations of 10.6 and 3.76 mg/l, respectively (based on a Williams’ multiple sequential test procedure, one-sided smaller, ¿ = 0.05), these effects were not considered as biologically relevant toxic effects based on the threshold (20%) defined by the DIN 38412-L33 norm1.
Therefore, the LOEC with respect to yield is 27.1 mg/l and the NOEC is 10.6 mg/l.
Reported statistics and error estimates:
Statistical analysis was performed with respect to the no observed effect concentrations. The statistical analysis was conducted with the software ToxRat® Standard Version 3.2.1.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In the control cultures: average specific growth rate of 1.72 (>0.92)/coefficient of variation of average specific growth rates: 0.6% (<=7%)/ mean coefficient of variation for section-by-section specific growth rates 14.7% (<=35%).
Conclusions:
The acute toxicity of 5-(Hydroxymethyl)furfural (CAS no. 67-47-0) to to the green alga Raphidocelis subcapitata was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of 5-(Hydroxymethyl)furfural to Raphidocelis subcapitata was 132 mg/l based on the measured concentration of the test item. The no observed effect concentration (NOErC) with respect to growth rate was 27.1 mg/l based on the measured concentration of the test item. The results of the test can be considered reliable without restriction.
Executive summary:

The growth inhibitory effects of 5-(Hydroxymethyl)furfural (CAS no. 67-47-0) to the green alga Raphidocelis subcapitata were investigated according to OECD guideline 201 over a period of 72 hours.

The test item 5-(Hydroxymethyl)furfural is solid, 85.4% pure and soluble in test water. The test solutions were prepared by appropriate dilutions of a stock solution in test water.

The nominal test item concentrations were 117, 58.5, 29.3, 14.6 and 7.32 mg/l (which correspond to 100, 50.0, 25.0, 12.5 and 6.25 mg/l of the main component). Three parallel test vessels were used

for each concentration of the test item and six vessels for the controls.

The concentrations of 5-(Hydroxymethyl)furfural in the test media were analyzed by HPLC with UV detection at the beginning and after 24, 48 and 72 hours of exposure. These analyses showed that the test concentrations were correctly dosed but decreased below 80% of the nominal value in the three lower concentrations over the 72 hours test period. Therefore, the effective concentrations ECx

were assessed based on the geometric mean of the measured concentrations of the test item over the entire 72 hours test period, which were 122, 59.7, 27.1, 10.6 and 3.76 mg/l.

With respect to the endpoint average specific growth rate, the following effects were observed compared to the control: 47% at 122 mg/l and 19% at 59.7 mg/l. No biologically relevant effects were observed at the three lower test concentrations.

With respect to the endpoint yield, the following effects were observed compared to the control: 92% at 122 mg/l, 63% at 59.7 mg/l, 24% at 27.1 mg/l. No biologically relevant effects were observed at the two lower test concentrations The results of the average specific growth rate and yield inhibition of 5-(Hydroxymethyl)furfural to the green alga Raphidocelis subcapitata are summarized in the following table based on the geometric mean of the measured concentrations (GMMC) of the test item:

Parameter (0-72 h)

 Growth rate [mg/l]

 Yield [mg/l]

 EC50

 132

 45.5

 EC10

 39.2

 16.2

 LOEC

59.7

 27.1

 NOEC

  27.1

  10.6

The 72 h EC50 value based on average specific growth rate of 5-(Hydroxymethyl)furfural (CAS no. 67-47-0) on the green alga Raphidocelis subcapitata was 132 mg/l and the EC10 was 39.2 mg/l based on the measured concentration of the test item.

All validity criteria were fulfilled.