Reaction products of N-Phenyl-1-Naphthylamine with Nonene, branched

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-01 - 2021-09-16

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test substance is poorly water soluble, therefore a water accommodated fractions (WAF) was prepared using a liquid-liquid saturator technique [Ref. 4, 6, 7]. Two glass aspirator bottles (2-L) with a bottom side-outlet attached to a stopcock were filled with 2- L test medium. For the saturation interval and for the last interval one glass aspirator bottle was sufficient. The medium was stirred at approx. 100 rpm and then 198-μL (considering density = 1.009 g/cm³) of the test substance was pipetted carefully on the water surface. The bottle was closed, and the solution was stirred on a magnetic plate for approximately 3 days. The stirring was slow (approx. 100 rpm) so that no vortex formed. The undissolved test substance remained on the surface and the required volume of the saturated solution (approx. 1.3-L) was removed from the bottom of each bottle. The removed test solution from each bottle was
merged in a 3L glass beaker were indicated.
The aqueous fraction was visually colorless and clear, and no undissolved test substance was visible. Nevertheless, the control and test substance group showed a positive Tyndall effect. The
positive Tyndall effect was seen equally in the control and test solutions, so this is probably due to the prolonged stirring and not due to presence of undissolved the test substance.
The control test solutions were treated in the same way. Fresh test solutions were prepared daily. The test vessels were saturated with test solution for approx. 1 day before the start of
exposure and renewal. All test solutions were visibly clear over the exposure period.

analysis:
The given water solubility is 0.7 μ/L ± 0.5 μg/L. In test media, the detectable concentrations in fresh prepared samples were significantly lower (approx. 0.03-0.18 μg/L. OECD guidance acknowledges that differences in solubility between pure water and aqueous test media (containing nutrient salts) are not unusual. All reasonable efforts were taken to produce a water
accommodated fraction (water saturated fraction) of the test substance according to guidance in OECD 23 and the aqueous fraction of the test solution, after separation of the undissolved
material, is considered analogous to a saturated solution of the test substance. No higher concentration of the test substance in test medium could be achieved.
For an evaluation of the results the overall geometric mean measured concentrations should be used.

Test groups: 0 (control) and 100 mg/L as nominal concentrations based on testsubstance density without correction for purity or content. 0 (control) and 0.0147 μg/L as overall geometric mean measured concentration.

Photoperiod: Continuous Culture medium: Synthetic fresh water (20X AAP).
The test medium (20X AAP medium) was prepared as described in OECD Guideline for Testing of Chemicals, No. 221 Lemna

Test organisms (species):

Lemna gibba

Details on test organisms:

Age (on study day 0) An inoculum culture of Lemna gibba (8 days old at 24 °C 2°) is used to initiate the test (study day 0).
Supplier: The stock culture was obtained from BASF SE, Agricultural Solutions (previous name Crop Protection) Ecology and Environmental Analytics, Speyerer Strasse
2, 67117 Limburgerhof, Germany.
Culture conditions: A culture is maintained continuously at the test facility. Before the exposure is started an inoculum culture is prepared with sufficient colonies of Lemna transferred aseptically into fresh sterile 20X AAP medium. The inoculum culture is incubated under test conditions for 8 days prior to test initiation. Reference substance testing: In order to verify that the Lemna cultures are responding normally to toxic stress, tests with a reference substance (3,5 Dichlorphenol) are conducted. Reference substance tests are conducted according to OECD 221 guidelines
and in accordance with GLP, but without a GLP status. The results from the refe rence substance test are compared to 3,5 Dichlorphenol EC50 values published in OECD Guideline [Ref 1], which represent the typical response range for the Lemna species tested. The EC50(7d) of the reference substance 3,5 Dichlorphenol was 7.78 mg/L.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24 ±2°C

Nominal and measured concentrations:

Concentration control analysis:
The given water solubility is 0.7 μ/L ± 0.5 μg/L. In test media, the detectable concentrations in fresh prepared samples were significantly lower (approx. 0.03-0.18 μg/L. OECD guidance acknowledges that differences in solubility between pure water and aqueous test media (containing nutrient salts) are not unusual. All reasonable efforts were taken to produce a water
accommodated fraction (water saturated fraction) of the test substance according to guidance in OECD 23 and the aqueous fraction of the test solution, after separation of the undissolved
material, is considered analogous to a saturated solution of the test substance. No higher concentration of the test substance in test medium could be achieved.
For an evaluation of the results the overall geometric mean measured concentrations should be used.

Details on test conditions:

EXPERIMENTAL PROCEDURE
Test duration: 7 days
Test vessels: Glass petri dishes covered with glass plates (nominal volume 300 mL; colorless glass with a minimum water depth of 20 mm)
Test medium: 20X AAP medium (see section 3.3.2)
Test volume: 160 mL
Initial colonies: 10 fronds/ test vessel at the start of exposure
Test chamber: Infors HT Multitron Pro controlled climate cabinet.
Test temperature: 24.3 – 24.5 °C
In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels.
Illumination: Artificial light, type universal white, permanent illumination. To minimize the potential effect of slight variations in illumination, the test vessels were rearranged daily.


Test initiation and maintainance
Colonies consisting of 3 to 4 visible fronds were transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions. Each test vessel should
contain a total of 10 fronds. The number of fronds and colonies were the same in each test vessel. The test vessels were impartially distributed in an incubator and with continuous
illumination. Vessel positions were altered each day. The temperature was monitored continuous within the incubator and instantaneous in a separate vessel filled with deionized
water. At the end of the test, the pH was measured in all replicates of the control and treatment group; the pH at test initiation was measured in the respective bulk solution.
Throughout the test, the appearance of the test solutions and dissolution behavior of the test substance was observed and recorded daily in old and new test solutions.
Light intensity: 7122–8069 lux (mean: 7413 lux, coefficient of variation: 4.05%) at a wavelength of 400 - 700 nm.
Route of exposure: Semi-static exposure via the test medium.
Reason: In order to insure constant exposure conditions this study was conducted as a static-renewal exposure. The renewal was done daily.
Randomization: The test vessels were placed in the test chamber according to a randomization plan prepared by using a program of the
laboratory data evaluation group of the testing facility. The test vessels were rearranged daily.
Test endpoint parameter: Growth as frond number and weight.
Test parameter: The frond number in the test vessels were counted at the start of exposure, at day 2, day 4 and at test termination (day 7). Dry weight was determined at the start of exposure in a comparable colony and at the end of exposure in all replicates.

Frond number
The frond number in the test vessels were counted at the start of the test, at day 2, day 4 and at the end of exposure (day 7) in each replicate. Every frond visibly projecting beyond the edge
of the parent frond was counted. Observations on the appearance of the fronds included necrosis, chlorosis, changes in plant size or shape and root growth were documented.

Dry weight
The biomass based on dry weight was determined at the start of exposure from a sample of the inoculum culture representative of what was used to begin the test, and at the end of the
test with the plant material from each treatment and control vessel. All colonies were collected from each of the test vessels and rinsed with deionized water. They were blotted to remove
excess water and then dried at approx. 60°C for 4 days to a constant weight. Any root fragments were included.

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 0.015 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 0.015 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.015 µg/L

95% CI:

>

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.015 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.015 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.015 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Test results: The duckweed population in the control vessels showed exponential growth, increasing from 10 fronds per vessel to an average of 246 fronds per vessel after 7 days (corresponding to a 24.6x multiplication). The dry weight increased from 1.4 mg (sample of the inoculum culture at test initiation) to an average of 38.3 mg per vessel in the control at test termination.
No toxic effects were observed in the saturated solutions with a loading rate of 100 mg/L up to the solubility limit of the test substance.
Since the measured concentrations deviated markedly from the nominal concentrations, the effect concentration, which is based on the overall geometric mean measured concentrations should be preferably used for the evaluation of the test substance.

Validity criteria fulfilled:

yes

Conclusions:

Percent growth inhibition relative to the control was calculated for the limit test concentration based upon growth rates and final yield for the parameters frond number and plant dry weight.
The 7-day ErC50 values determined based on overall geometric mean measured concentrations in this toxicity study were:
concentrations in this toxicity study were:
Average specific growth rate >0.0147 µg/L based ion frond number and dry weight
ErC50 (95% CL)
Yield
EyC50 (95% CL) >0.0147 µg/L based ion frond number and dry weight

Since the measured concentrations deviated markedly from the nominal concentrations, the effect concentration, which is based on the mean measured concentrations should be preferably used for the evaluation of the test substance.
The toxicity results presented here are consistent with the results from preliminary tests.
The results in this study are consistent with all validity criteria and the test is valid according tothe guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

Description of key information

In an OECD 221 with Lemna gibba the aquatic toxicty of the test item was evaluated towards aquatic plants. In a semi-static appoach with a daily water exchange over 7 day Leman gibba was exposed towards the test item. Since the test substance is poorly water soluble,  a water accommodated fractions (WAF) was prepared using a liquid-liquid saturator technique. The solubility of the test item is in the range of 0.0147 µg/L. Within this concentration, no toxic effects on Lemna gibba could be observed.

ErC10: > 0.0147 µg/L average specific growth rate based on frond mumber and dry weight

ErC50: > 0.0147 µg/L average specific growth rate based on frond mumber and dry weight

Key value for chemical safety assessment

EC50 for freshwater plants:

0.015 µg/L

EC10 or NOEC for freshwater plants:

0.015 µg/L