Condensation products of phenol and salicylaldehyde

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:

29 June 2016 to 01 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-2-1 The guidelines related to the study reports for the registration application of pesticide

Version / remarks:

Ref. No. 12-Nousan-8147 on 24 November 2000 & Ref. No.13-Seisan-3986 on 10 October 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Since analytical measurement was performed by TOC analysis and therefore only the sum of the carbon content of the Test Material was measured this way (and this did not change by the end of the test, independently from the stability), measurement was sufficient only at the beginning of the renewal periods to justify that the Test Material was present in the test solution, since more accurate data can not be provided by TOC. Therefore concentration of the Test Material was measured at all of the test concentration at the start of each renewal period. Sample from the control was taken for analysis at the start of the test.

Vehicle:

no

Details on test solutions:

- Because the test material is a polymer, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. Saturated Test Material solutions (mg/L nominal loading rates WAFs) were prepared individually by dispersing/dissolving the needed amount of test material into the test medium (OECD Medium) two days before the start of the experiment. These solutions were shaken for about 24 hours at approximately 30 °C and then were equilibrated for about 24 hours at approximately 20 °C. The non-dissolved test materials were removed by filtration through a fine (0.22 μm) filter to give the 100 % saturated solutions.
- Taking into account that the stability of the test material was not available, the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to the treatments, at each of the renewal periods test solution was prepared by the method described above.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Age: Less than 24 h old at the beginning of the test.
- Feeding during test: No

ACCLIMATION
- Acclimation period: There was no acclimatisation because the water used was similar to the culture water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

249 mg/L (as CaCO3)

Test temperature:

20.1 – 20.4 °C

pH:

7.55 – 7.90

Dissolved oxygen:

7.1 – 8.5 mg/L

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25, 50 and 100 mg/L nominal loading rate WAF.
Measured: 2.01, 8.77, 21.3, 48.2 and 88.5 mg/L
Biological results are based on the nominal test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker
- Renewal rate of test solution: Every 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: At least 5 mL test solution/animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
- Separate stock solutions of individual trace elements were first prepared in deionised water. The medium was prepared of these different stock solutions, so it contained all trace elements (combined solution): CaCl2×2 H2O 11.76 g/L 25 mL/L, MgSO4×7 H2O 4.93 g/L 25 ml/L, KCl 0.23 g/L 25 mL/L and NaHCO3 2.59 g/L 25 mL/L.
- Intervals of water quality measurement: The water temperature, the oxygen concentrations and pH of the controls and the test solutions were measured at the beginning and at the end of each renewal period.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: The test was carried out in 16-hour light and 8-hour dark cycle.

EFFECT PARAMETERS MEASURED
- The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
- The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10 and 100 mg/L nominal loading rates WAF
- A concentration range-finding test was conducted to determine the approximate toxicity of the Test Item so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions.
- Results used to determine the conditions for the definitive study: Yes, because significant immobility was observed at the highest examined concentration level during the Preliminary Range-Finding Test, five test concentrations in a geometric series (factor 2.0) plus a control were used in the Main Test under semi-static conditions. The concentrations in the Definitive Test are based on the results of the Preliminary Range-Finding Test and were: 6.25, 12.5, 25, 50 and 100 mg/L nominal loading rates WAFs.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

36.05 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits: 33.86 – 38.25 mg/L nominal loading rate WAF

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

100 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

25 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

50 other: mg/L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

IMMOBILISATION
- The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour (Table 1).
- At the Test Material concentration of 25 mg/L nominal loading rates WAF 10 % immobilisations were observed which is not above the acceptable control level furthermore statistically significantly not different from the Control therefore it was determined as the NOELR.

Results with reference substance (positive control):

- For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
- The date of the last study with reference material Potassium dichromate was: 12-13 January 2016.
- The 24h EC50: 1.16 mg/L, (95 % confidence limits: 1.07 – 1.25 mg/L)

Reported statistics and error estimates:

The 24 hours EC50 could not be calculated (due to 25 % of immobilisation in the highest concentration). The 48 hours EL50 values were calculated using Probit analysis by TOXSTAT software. The 48h EL100 value of the Test Material was determined from the raw data. For the determination of the LOELR and NOELR, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Validity

- There were no immobilised animals in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Table 1: Number and percentage of immobilised animals

Nominal Concentrations (mg/L nominal loading rates WAFs)	Number of treated animals	Immobilised Animals
		24 Hours		48 %
		Number	%	Number	%
Control	20	0	0	0	0
6.25	20	0	0	0	0
12.50	20	0	0	0	0
25.00	20	0	0	2	10
50.00	20	4	20	19	95*
100.00	20	5	25*	20	100*

*: Statistically significantly different compared to the control values (Dunnett’s Test; α = 0.05).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the 48 h EL50 value was 36.05 mg/L nominal loading rate WAF (95 % confidence limits: 33.86 – 38.25 mg/L nominal loading rate WAF). The 48 h EL100 value was 100 mg/L nominal loading rate WAF, the 48h No-Observed Effect Loading Rate (NOELR) was 25 mg/L nominal loading rate WAF and the 48h Lowest Observed Effect Loading Rate (LOELR) was 50 mg/L nominal loading rate WAF.

Executive summary:

The acute toxicity of the test material was assessed with an Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system. The test was conducted in accordance with the standardised guidelines OECD 202, EU Method C.2 and OCSPP 850.1010, under GLP conditions.

Based on the performed preliminary range-finding test five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment.

The nominal concentrations of Test Material used in the main experiment were: 6.25, 12.5, 25, 50 and 100 mg/L nominal loading rates WAFs.

Since analytical measurement was performed by TOC analysis and therefore only the sum of the carbon content of the Test Material was measured this way (and this was not change by the end of the test, independently from the stability), measurement was sufficient only at the beginning of the renewal periods to justify that the Test Material is present in the test solution, since more accurate data can not be provided by TOC. Therefore biological results are based on the nominal test material concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test and for the control groups. The 24 hours EL50 could not be calculated due to 25 % of immobilisation in the highest concentration. The 48 hours EL50 values and their confidence intervals were calculated using Probit analysis by TOXSTAT software. The 48 h EL100 value of the Test Material was determined from the raw data. For the determination of the LOELR and NOELR, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

All validity criteria were met during this study.

Under the conditions of this study the 48 h EL50 value was 36.05 mg/L nominal loading rate WAF (95 % confidence limits: 33.86 – 38.25 mg/L nominal loading rate WAF). The 48 h EL100 value was 100 mg/L nominal loading rate WAF, the 48 h No-Observed Effect Loading Rate (NOELR) was 25 mg/L nominal loading rate WAF and the 48 h Lowest Observed Effect Loading Rate (LOELR) was 50 mg/L nominal loading rate WAF.

Description of key information

Under the conditions of this study the 48h EL50 value was 36.05 mg/L nominal loading rate WAF (95 % confidence limits: 33.86 – 38.25 mg/L nominal loading rate WAF). The 48h EL100 value was 100 mg/L nominal loading rate WAF, the 48h No-Observed Effect Loading Rate (NOELR) was 25 mg/L nominal loading rate WAF and the 48h Lowest Observed Effect Loading Rate (LOELR) was 50 mg/L nominal loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

36.05 mg/L

Additional information

The acute toxicity of the test material was assessed with an Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system. The test was conducted in accordance with the standardised guidelines OECD 202, EU Method C.2 and OCSPP 850.1010, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based on the performed preliminary range-finding test five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment. The nominal concentrations of Test Material used in the main experiment were: 6.25, 12.5, 25, 50 and 100 mg/L nominal loading rates WAFs.

Since analytical measurement was performed by TOC analysis and therefore only the sum of the carbon content of the Test Material was measured this way (and this was not change by the end of the test, independently from the stability), measurement was sufficient only at the beginning of the renewal periods to justify that the Test Material is present in the test solution, since more accurate data can not be provided by TOC. Therefore biological results are based on the nominal test material concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test and for the control groups. The 24 hours EL50 could not be calculated due to 25 % of immobilisation in the highest concentration. The 48 hours EL50 values and their confidence intervals were calculated using Probit analysis by TOXSTAT software. The 48h EL100 value of the Test Material was determined from the raw data. For the determination of the LOELR and NOELR, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

All validity criteria were met during this study.

Under the conditions of this study the 48h EL50 value was 36.05 mg/L nominal loading rate WAF (95 % confidence limits: 33.86 – 38.25 mg/L nominal loading rate WAF). The 48h EL100 value was 100 mg/L nominal loading rate WAF, the 48h No-Observed Effect Loading Rate (NOELR) was 25 mg/L nominal loading rate WAF and the 48h Lowest Observed Effect Loading Rate (LOELR) was 50 mg/L nominal loading rate WAF.