Reaction product of salicylaldehyde and formaldehyde and phenol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May 2012 to 16 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Daphnia sp., Acute Immobilization Test stipulated in title "Testing Methods for New Chemical Substances" of Japan (March 31, 2011; No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare

Version / remarks:

March 29, 2011, No.5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 23 (Guidance Document on Aquatic Toxicity Testing of Diificult Substances and Mixtures)

Version / remarks:

2000

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Concentrations were measured at the start and end of exposure
- Sampling method: Another solution was sampled separately from the container for preparation (at the start of exposure). The mixed solution taken out with equal volume from the middle layer of the test solution in test vessels in each test level (at the end of exposure). Approximately 10 mL was taken at all test levels.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The ground test material sample was weighed and dissolved in acetone to prepare 10000 mg/L of the test material solution. The solution was accordingly diluted with acetone to prepare 100 times as much as each test concentration. The test material solution of each concentration was added into the preparation container, and inner of the container was coated with the test material by evaporating the acetone under a stream of nitrogen. To remove acetone by volatilisation completely, the container was heated on a hot plate (approximately 40 °C) for approximately 10 minutes. Dilution water was added into each container to prepare each test concentration (nominal concentration) and stirred for 24 hours at approximately 20 °C. At the end of the stirring period the suspension was filtered with a glass fibre filter (GB-140, 0.4 µm pore size, Toyo Rosin) by suction. The filtrate was stirred for 30 minutes at about 20 °C to recover dissolved oxygen concentration decreased at the suction filtration, and then used as the test solution. These test solutions were divided into each test vessel.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: The University of Sheffield (Sheffield S10 2UQ, United Kingdom)
- Age: Less than 24-hour-old daplmids
- Feeding during test: no

CULTURE CONDITIONS
Young daphnids produced by parents that were cultured in the testing laboratory were used. The parents to obtain young daphnids were bred in the same quality of water (ASTM medium), water temperature (20 ± 1 °C) and photoperiod (16-hour light/8-hour dark) as used in the test. The parent animals used for the test were same lot and bred for more than 14 days, and their age and survival rate were 20-day old and 100%, respectively. Chlorella vulgaris of 0.1-0.2 mgC (Organic carbon content)/day per Daphnia was fed to the parents once a day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

157.3 mg CaCO3/L

Test temperature:

19.8 - 20.2 °C

pH:

8.1 - 8.3

Dissolved oxygen:

8.6 - 8.8 mg/L

Nominal and measured concentrations:

Nominal loading: 6.3, 13, 25, 50 and 100 mg/L
Geometric Mean Measured: 6.3, 13, 25, 51 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beaker
- Type: covered with a transparent plastic lid
- Material, size, headspace, fill volume: 100 mL/test vessel
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A reconstituted water (ASTM E 729; 2007) was used for dilution water. Selenium and Vitamin B12 were additionally put into it at 1 µg/L. The content constituents are: NaHCO3 192 mg/L, CaSO4.2H2O 120 mg/L, MgSO4.7H2O 246 mg/L, KCl 8 mg/L, Na2SeO3 0.00219 mg/L and Vitamin B12 0.001 mg/L.
- Intervals of water quality measurement: Dissolved oxygen concentration, pH and water temperature were measured at the start and end of exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Artificial light of white fluorescent lamp, 16-hour light/8-hour dark

EFFECT PARAMETERS MEASURED:
- Observations of immobility and symptoms of test organisms were conducted at 24 and 48 hours after exposure. Daphnids were considered immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessel.
- Appearance of the test solution was observed at the start and end of exposure.

CONCENTRATIONS
- Test concentrations with a geometric series with a factor of 2.0 were used
- The test concentrations and the geometric factor were decided from the results of preliminary studies.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

IMMOBILITY
- The minimum concentration causing 100% immobility at 48 hours was 50 mg/L. Maximum concentration causing no immobility at 48 hours was 13 mg/L. Immobility in the control during exposure was 0%, which meets the criterion for the validity of the test (i.e. not more than 10%).

OBSERVED ABNORMAL RESPONSE
- In the exposure level, lethargic, immobilisation and reduced activity were observed. In the control, no abnormal response (discolour of body, trapping at the surface of the water and so on), which met the criterion for the validity of the test (i.e. not more than 10%), was observed during exposure.

APPEARANCE OF THE TEST SOLUTION
- The test solutions in the 6.3-25 mg/L exposure levels were colourless and clear, and those in the other exposure levels of 50 and 100 mg/L were slightly red and clear in a concentration-dependent manner at the start and end of exposure. Appearance of test solution at the end of exposure was the same as that at the start of exposure. The test solution in the control was colourless and clear at the start and end of exposure.

CONDITION OF THE TEST SOLUTIONS
- The measured values of dissolved oxygen concentration, pH and temperature of the test solutions during exposure ranged from 8.6 to 8.8 mg/L, from 8.1 to 8.3 and from 19.8 to 20.2°C, respectively. The measured values of dissolved oxygen concentration met the criterion for the study validity (more than 13 mg/L at the end of exposure).

CONCENTRATION OF TEST MATERIAL IN THE TEST SOLUTIONS
- The measured concentrations of the test material in the test solutions at the start of exposure were 6.5-100 mg/L which were 101-108 % of the nominal concentration, and those at the end of exposure were 6.1-97 mg/L which were 97-101 % of the concentrations at the start of exposure.

Results with reference substance (positive control):

- Results with reference substance valid? yes, the value was within the stipulated range (mean ± 2 S.D.) [mean ± S.D.: 0.83 ± 0.18 mg/L (n=9)] to background data in this laboratory.
- ECx: 48-hour EC50 = 1.2 mg/L

Reported statistics and error estimates:

The 48-hour EC50 value was calculated by Binomial test.
The EC50 was estimated by using Computer Program (running on Microsoft software "Excel") developed by our laboratory.
The results of the study were estimated by nominal concentration, because the measured concentrations of the test item in the test solution were within the range of ± 20 % of that at the start of exposure.

Table 1: Immobility in the Study

Nominal Concentration (mg/L)		Immobility (%)
		24 Hours		48 Hours
		Replicate	Test Level	Replicate	Test Level
Control	A	0	0	0	0
	B	0		0
	C	0		0
	D	0		0
6.3	A	0	0	0	0
	B	0		0
	C	0		0
	D	0		0
13	A	0	0	0	0
	B	0		0
	C	0		0
	D	0		0
25	A	0	0	100	95
	B	0		80
	C	0		100
	D	0		100
50	A	40	25	100	100
	B	0		100
	C	40		100
	D	20		100
100	A	100	100	100	100
	B	100		100
	C	100		100
	D	100		100

Table 2: Measured Concentrations of Test Material in the Test Solutions

Nominal Concentration (mg/L)	Measured Concentration (mg/L) (Percentage of measured concentration versus nominal concentration %)
	Start of the Test	End of the Test	Geometric Mean
Control	n.d.	n.d.	-
6.3	6.5 (103)	6.1 (97)	6.3 (100)
13	14 (108)	13 (99)	13 (104)
25	25 (101)	24 (98)	25 (99)
50	52 (104)	50 (101)	51 (102)
100	100 (104)	97 (97)	100 (100)

n.d.: <0.200 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48-hour EC50 was 19 mg/L.

Executive summary:

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202 and the Japanese testing methods for new chemical substances, under GLP conditions.

An acute immobilisation study was performed with Daphnia magna using five test concentrations of 100, 50, 25, 13 and 6.3 mg/L as nominal loading concentrations (a geometric series with a factor of 2.0) and a control. The daphnia were exposed to the test material for 48 hours in a static test regime.

The test material sample dissolved in acetone was added to each preparation container and coated to the inner of the container by evaporating acetone, the dilution water was added to the container to prepare each nominal concentration. They were stirred for 24 hours and then filtered with a glass fibre filter to produce each test solution.

This study was conducted in order to confirm the effect of the test material on the test organisms below the solubility of the test material in dilution water. As a result, the 48-hour EC50 was 19 mg/L. The minimum concentration of the test material causing 100% immobility at 48 hours was 50 mg/L, the maximum concentration of the test material causing no immobility at 48 hours was 13 mg/L.

The measured concentrations of the test material in the test solution were within the range of ± 20% of those at the start of exposure. The environmental conditions were within the suitable range; therefore, it was concluded that this study complied with the applied test guidelines.

Under the conditions of this study, the 48-hour EC50 was 19 mg/L.

Description of key information

Under the conditions of this study, the 48-hour EC50 was 19 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

19 mg/L

Additional information

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202 and the Japanese testing methods for new chemical substances, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

An acute immobilisation study was performed with Daphnia magna using five test concentrations of 100, 50, 25, 13 and 6.3 mg/L as nominal loading concentrations (a geometric series with a factor of 2.0) and a control. The daphnia were exposed to the test material for 48 hours in a static test regime.

The test material sample dissolved in acetone was added to each preparation container and coated to the inner of the container by evaporating acetone, the dilution water was added to the container to prepare each nominal concentration. They were stirred for 24 hours and then filtered with a glass fibre filter to produce each test solution.

This study was conducted in order to confirm the effect of the test material on the test organisms below the solubility of the test material in dilution water. As a result, the 48-hour EC50 was 19 mg/L. The minimum concentration of the test material causing 100% immobility at 48 hours was 50 mg/L, the maximum concentration of the test material causing no immobility at 48 hours was 13 mg/L.

The measured concentrations of the test material in the test solution were within the range of ± 20% of those at the start of exposure. The environmental conditions were within the suitable range; therefore, it was concluded that this study complied with the applied test guidelines.

Under the conditions of this study, the 48-hour EC50 was 19 mg/L.