Phenol, 4,4′-(1-methylethylidene)bis-, polymer with 2-(chloromethyl)oxirane and N1,N1-diethyl-1,3-propanediamine

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:

23 March 2018 - 01 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006; Annex 5 corrected 28 July 2011.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

DEPDA adduct

Cas Number:

123739-10-6

IUPAC Name:

DEPDA adduct

Test material form:

solid

Details on test material:

- Physical appearance: dark amber solid
- Storage conditions: at room temperature

Specific details on test material used for the study:

- No correction factor for purity was required and therefore not applied.
- The test material is irritant/corrosive.
- Stability at higher temperatures: yes, overnight at maximum 45°C or for maximum 1 hour at maximum 80°C.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For determination of test concentrations in the final test, single samples for possible analysis were taken from the stock solutions, all test concentrations and the controls according to the schedule below:
Frequency: at t=0 h, t=48 h and t=72 h
Volume: 20 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the test facility

Test solutions

Vehicle:

yes

Remarks:

Tetrahydrofuraan

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Range-finding and final study: Stock solutions were prepared in tetrahydrofuran (THF, VWR International, Batch 1820907) at nominal concentrations ranging between 0.030 and 30 mg/mL. Preparation of the stock solutions started with the highest stock solution of 30 mg/mL, applying 10 minutes of ultrasonic treatment to ensure maximum dissolution of the test item in medium. The lower stock solutions were prepared separately by proportional dilution of the highest stock solution in THF. For the preparation of each test concentration, 100 µL of a stock solution was added to 1 L of test medium resulting in test concentrations that were a factor of 10,000 lower than the stock concentrations.
- Evidence of undissolved material: stock solutions appeared to be light brown/slightly hazy and contained small undissolved particles of which the nature is unknown. Test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Blank-control (test medium without test item or other additives) and solvent-control (test medium without test item but with 0.1 mL THF/L)

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Pre-culture: 4 days before the start of the (final) test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1, according to NPR 6505
- Pre-culture medium and test medium: M2, according to OECD201

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 24°C

pH:

At t=0 h: 7.9-8.1
At t=72 h: 7.9-8.2

Nominal and measured concentrations:

Nominal concentrations: 0.96, 3.0, 9.6, 30, 96 and 300 µg/L.
Measured concentrations: all test concentrations were below the limit of quantification (1 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, capped (aluminium caps) and perforated for ventilation, containing 50 mL of test solution
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 145 x 10^4 cells/mL (blank control) and 151 x 10^4 cells/mL (solvent control)
- 3 replicates of each test concentration,
- 6 replicates of the control,
- 2 extra replicates of each test group for sampling purposes;
- 2 or 3 replicates of each test concentration without algae.
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Culture medium different from test medium: yes, M1
- Intervals of water quality measurement: pH: at the beginning and at the end of the test. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 92 to 94 µE.m-2.s-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Other: At the end of the final test microscopic observations were performed on the nominally spiked concentration of 96 µg/L to observe for any abnormal appearance of the algae.

LIMIT/RANGE-FINDING TEST:
- A range-finding study was performed using concentrations of 0.0030, 0.030, 0.30 and 3.0 mg/L. Results showed that growth was inhibited by 33% and 95% at concentrations of 0.30 and 0.3 mg/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (July 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

246 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%-CI: 195 - 337 µg/L.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

30 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analytical results: since the test solutions were prepared at nominal concentrations below the LOQ (i.e. 1.0 mg/L) of the validated method, no quantifiable concentrations could be analysed. The test item concentration in the stock solutions prepared in THF could not be quantified either because of undissolved particles. As no alternative solvent which provided sufficient solubility nor analytical method was available, the effect concentrations were based on nominal spiked concentrations.

Results of the final test:
- Statistically significant growth rate inhibition of respectively 34% and 52% was recorded at nominal concentrations of 96 and 300 µg/L at the end of the test.
- No significant difference was present between the blank and solvent control and, therefore, measurements of both controls were pooled.
- Abnormalities observed: no, microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 96 µg/L when compared to the control.
- The test conditions were within the limits prescribed by the study plan and the test guidelines.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Test concentrations: 0, 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L.
- The observed 72h-ERC50 for the algal culture tested corresponds with the historical range of the test facility.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in both the negative and solvent control (pooled) revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsch-t-test with Bonferroni-Holm correction, α=0.05, one-sided, smaller) or reduction of yield (Williams Multiple Sequential t-Test, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominally spiked concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Table 1 Growth Rate and Percentage Inhibition for the Total Test Period

INCA 460: DEPDA Adduct Nominal conc. (µg/L)	Mean	Std. Dev.	n	%Inhibition
Pooled control	1.664	0.0315	12
0.96	1.655	0.0132	3	0.54
3.0	1.689	0.0140	3	-1.5
9.6	1.650	0.0296	3	0.89
30	1.654	0.0591	3	0.64
96	1.104	0.1024	3	34*
300	0.793	0.1741	3	52*

* - effect was statistically significant.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks'

Conclusions:

Based on an algae toxicity study, performed according to OECD guideline 201 and GLP, the 72h-EC50 for growth rate inhibition of INCA 460: DEPDA Adduct towards Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was 246 µg/L (95% confidence interval ranging from 195 to 337 µg/L).