12 Emergence Test with Chironomids (OECD TG 219) – TiO 2
12.5.6 Total exposure duration The exposure period was 28 days. The exposure period was 28 days
• P25: April 21 - May 19, 2010
• NM-101: May 27 - June 24, 2010
No post-exposure observation period was performed.
12.5.7 Test conditions
Hardness: Test start 110 – 150 mg/L as CaCO3 equivalents (de-manded threshold value of 400 mg/L as CaCO3 equi-valents)
Test end: 100 – 120 mg/L as CaCO3 equivalents in the controls and 210 mg/L as CaCO3 equivalents in one representative replicate of the highest test concentration P25
Test temperature: 20.3°C (permitted range: 20 ± 2°C)
pH: 7.9 – 8.4 (permitted range: pH 6 – 9)
Dissolved oxygen: About 100% at test start and test end (demanded threshold value: 60%)
Ammonia: Test start: 0.8 - 1.0 (control); 0.2 (highest test concent-ration)
Test end: 8.0 - 10.2 (control); 9.2 (highest test concent-ration)
Nominal concentrations: The nominal concentrations in the test containers with TiO2 nanoparticles were 15, 23, 39, 63, and 100 mg test item/L.
Details on test conditions: The light intensity was measured using an illuminance meter (MINOLTA) with photometric sensor in lux. With 748 – 850 lux the permitted range of about 500 - 1000 lux was kept.
Hardness: At test start 130 – 150 mg/L CaCO3 equivalents in the control and 140 mg/L CaCO3 equivalents in one repre-sentative replicate of the highest test concentration (demanded threshold value of 400 mg/L as CaCO3 equivalents)
Test end: 150 – 170 mg/L CaCO3 equivalents in the control and 170 mg/L CaCO3 equivalents in one repre-sentative replicate of the highest test concentration.
NM-101
Test temperature: 20.3 °C -20.5°C (permitted range: 20 ± 2 °C)
pH: 7.8 – 8.7 (permitted range: pH 6 – 9)
Dissolved oxygen: About 100% at test start and test end (demanded threshold value: 60%)
Ammonia: Test start: 0.5 - 0.9 (control); 0.7 (highest test concent-ration)
Test end: 0.1 - 7.5 (control); 0.6 (highest test concent-ration)
Nominal concentrations: The nominal concentrations in the test containers with TiO2 nanoparticles were 15, 23, 39, 63, and 100 mg test item/L.
Details on test conditions: The light intensity was measured using an illuminance meter (MINOLTA) with photometric sensor in lux. With 771 – 826 lux the permitted range of about 500 – 1000 lux was kept.
According to the guideline a test with a reference substance is not compulsory.
However, 2-chloracetamid was tested in a sediment-water chironomid toxicity test using spiked sediment (OECD 218).
Reference substance:
12.5.8 Other information on materials and methods
The control consists of sediment, tap water and chironomids. Four replicates per control were conducted.
Control treatment
Data evaluation:
Statistical method
Numerical values in this report are frequently rounded to a smaller degree of precision (num-ber of digits) than used in the actual calculation. Minor differences in the results obtained from calculations with the rounded values compared to the values obtained with higher preci-sion values are possible. They are, however, well within the limits of the experimental accu-racy and thus of no practical concern.
Statistical calculations:
The number of emerged males and females were determined. The results of the listed bio-logical parameters (total, males, females) were compared by a suitable test for multiple com-parisons with a control after testing variance homogeneity. All statistical tests were per-formed with the computer software ToxRat Professional version 2.10.4.1 (ToxRat® Solutions GmbH).
TetraMin® Hauptfutter powder was used for feeding the larvae. According to the guideline the food ration for the first 10 days was 0.25 – 0.5 mg TetraMin® /larvae/day, from day 10 on the food ration was increased to 0.5 – 1.0 mg TetraMin® /larvae/day.
Food
Round glass beakers (3L) were used as test vessels. The vessels were filled to a height of 2 cm with wet artificial sediment (corresponding to 370 g dry mass). The overlaying water was 8 cm high (ratio sediment:water about 1:4). The containers were covered with glass plates. After 10 days, emergence traps were placed on the test vessels, the glass plates re-mained on the emergence traps to avoid evaporation. Aeration of overlaying water was pro-vided through a glass pipette fixed 2-3 cm above the sediment layer (at least 1 bubble /second).
Test container
Sediment was placed into the test vessels. Four hundred millilitres of tap water was added and the sediment-water system was left under gentle aeration for several days prior to add-ing the test organisms. Batches of twenty larvae were placed into each vessel.
Test procedure
After incubation for 24 h, 500 mL of the freshly prepared stock dispersion of the nanoparti-cles was added. A further 100 mL of tap water was used to rinse the vessels containing the stock dispersions. To avoid separation of sediment ingredients during addition of test water
while water was poured onto it. The disc was removed immediately afterwards. Due to the large amount of treatment solution, the dispersion mixed while being added to the water col-umn. There was no further mixing to avoid disturbance of the sediment.
The test was carried out at 20°C ± 2°C and at 16 h photoperiod (500 –1000 lux). The expo-sure duration was 28 days. Development time and total number of fully emerged male and female midges were determined. Test vessels were observed daily for visual assessment of abnormal behaviour. Emergence was counted daily. After identification the midges were re-moved from the test vessel. At test end, the test vessels were observed for visible pupae that had failed to emerge.
12.6 Results 12.6.1 P25
(Raw data, chapter 21.5.1)
The zeta potential is presented in Zeta potential
Table 82. The three highest test concentrations were de-termined. No significant difference between the concentrations was observed.
Table 82: P25 - Test with chironomids: zeta potential.
Sample Zeta potential [mV]
39 mg/L -20.7
63 mg/L -18.8
100 mg/L -19.4
Particle size distribution is presented in Table 83. At day 0 a difference between the control and the samples containing TiO2 nanoparticles is observed at concentrations of 24 -
100 mg/L, where a large peak above 1000 nm was detected. The size of the peak increases with increasing concentration. It is assumed that the particles determined in the control origi-nate from the sediment. At day 1 no difference between the control and the vessels contain-ing TiO2 nanoparticles was observed. It is assumed that the agglomerates measured at day 0 were sedimented. Therefore, no further measurements were performed. By chemical analy-sis, Ti was detected in the aqueous phase at day 1 (see below).
Table 83: P25 - Test with chironomids: particle size distribution (spiked water).
Concentration [mg/L]
Z-Average
[nm] 1 PDI 2 Peak 1 [nm]
Peak 2 [nm]
Count rate 3 [kcps]
Measurement
position 4 Attenuation 5 Day 0
Control 1542 0.8 616 - 110 1.25 7
15 1101 0.5 737 - 162 4.65 7
24 2019 0.5 1070 - 184 4.65 6
39 2111 0.5 1208 - 79 4.65 5
63 2262 0.4 1364 - 32 1.25 4
100 2060 0.2 1659 - 486 1.05 6
Day 1
Control 2551 1 302 - 198 4.65 8
15 970 0.6 481 - 289 4.65 9
24 1377 0.8 633 - 148 4.65 8
39 1871 0.9 525 - 67 4.65 7
63 1482 0.8 550 - 166 4.65 9
100 1971 1 535 - 149 4.65 9
1 calculated value (cumulative mean); 2 increasing value indicates increasing polydispersity (maximum: 1); 3 best results with a count rate between 150 and 500 kilo counts per second (kcps); 4 measurement position in the mid-dle of the measuring cell; 5 indicator for turbidity (high values indicate low turbidity; maximum: 11); 6 1 mg/L sam-ples below quantification limit; 7 prepared from 10 mg/L samples with 3 min of ultrasonic treatment; 8 prepared from 10 mg/L samples with 30 min of ultrasonic treatment
Titanium concentrations in the overlaying water at several measuring dates are presented in Test item concentrations
Table 84. There was sedimentation of TiO2 nanoparticles resulting in Ti concentrations in the overlaying water below the detection limit at the end of the test. Information concerning the validity of the analyses is included in the annex. Due to the high background values (refer to Table 88 for NM-101) in this experimentTi was not analysed in the sediment.
Table 84: P25 - Test with chironomids: Ti concentration.
Sample
Control 15 mg/L 24 mg/L 39 mg/L 63 mg/L 100 mg/L Ti [mg/L] 9.0 1 14.4 1 23.4 1 37.8 1 59.9 1 Day 1
Water Replicate 1 [µg/L] 12.1 522.2 621.6 645.6 466.5 467.8 Replicate 2 [µg/L] 11.6 515.2 632.7 656.7 466.3 474.6
mean value [µg/L] 11.8 518.7 627.2 651.2 466.4 471.2
Day 7
Water Replicate 1 [µg/L] 8.9 18.6 23.7 34.9 18.1 32.7
Replicate 2 [µg/L] 8.6 18.2 23.0 34.3 16.0 36.1
mean value [µg/L] 8.8 18.4 23.4 34.6 17.1 34.4
Day 14
Water Replicate 1 [µg/L] 60.7 57.9 166.0 71.6 105.0 20.4
Replicate 2 [µg/L] 58.5 56.0 171.0 69.2 102.0 19.9
mean value [µg/L] 59.6 57.0 168.5 70.4 103.5 20.2
Day 28
Water Replicate 1 [µg/L] 2 2 2 2 2 2
Replicate 2 [µg/L] 2 2 2 2 2 2
MW [µg/L]
1 nominal values for Ti (calculated from TiO2 concentrations); 2 < LOQ/LOD
Summarised results are presented in Effects
Table 85 and Table 86.
No significant effect was observed.
The NOEC (no observed effect concentration) of the biological endpoints was ≥ 100 mg/L.
EC10, EC20, and EC50 values of the biological endpoints were all > 100 mg/L.
Neither physical nor pathological symptoms were obtained. All specimens gave the impres-sion of healthy condition. Only one emerged animal died, in the second concentration (24 mg/L) at day 18.
Physical/pathological symptoms and changes in behaviour
The results of emergence are presented in Emergence rate
Table 85.
No concentration/effect dependency on emergence rate due to P25 was detected. The NOEC for the tested species Chironomus riparius was found to be > 100 mg/L for males, females, and the combined sexes.
Table 85: P25 - Test with chironomids: emergence.
Emergence: number of individuals; emergence rate: % of introduced larvae; concentrations given as nominal values
Control 15 mg/L 24 mg/L 39 mg/L 63 mg/L 100 mg/L
Emerged midges [Ind.]
54 58 61 53 55 53
Emergence rate midges [%]
67.5 72.5 76.2 66.2 68.7 66.2
Emerged midges [males]
20 30 32 23 31 28
Emerged midges [females]
34 28 29 30 24 25
The results for development time and rate are presented as mean values ( Development time and rate
Table 86). No in-fluence on emergence due to P25 was detected.
Table 86: P25 - Test with chironomids: development time [d] and rate [1/d].
Concentrations given as nominal values.
Control 15 mg/L 24 mg/L 39 mg/L 63 mg/L 100 mg/L
Development time midges
18.5 18.5 17.5 17.5 17.2 18.5
Development rate midges
0.054 0.054 0.057 0.057 0.058 0.054
Development time males
17.5 17.5 16.4 16.4 16.4 16.9
Development rate males
0.057 0.057 0.061 0.061 0.061 0.059
Development time females
19.2 19.2 18.9 18.2 18.2 18.5
Development rate females
0.052 0.052 0.053 0.055 0.055 0.054
12.6.2 NM-101
(Raw data, chapter 21.5.2)The zeta potential is presented in Zeta potential
Table 87. The three highest test concentrations were de-termined. No significant difference between the concentrations was observed.
Table 87: Test with chironomids - NM-101: zeta potential.
Sample Zeta potential [mV]
39 mg/L -19.1
63 mg/L -17.7
100 mg/L -19.5
Ti concentrations in the overlaying water and in the sediment are presented in Test item concentrations
Table 88.
There was sedimentation of TiO2 nanoparticles resulting in decreasing Ti concentrations in the overlaying water. At test end nearly all of the TiO2 was determined in the sediment. The test concentrations were low compared to the background of Ti in the sediment. Due to the high background values of Ti in the sediment, calculated recovery of the added Ti is not very precise. At least at the two highest test concentrations, recovery was within the desired range.
Table 88: NM-101 - Test with chironomids: Ti concentrations.
Sample
Control 15 mg/L 24 mg/L 39 mg/L 63 mg/L 100 mg/L Ti [mg/L] 9.0 1 14.4 1 23.4 1 37.8 1 59.9 1 Day 1
Water Replicate 1 [µg/L] 6.6 825 1159 1626 1637 1310
Replicate 2 [µg/L] 6.9 824 1167 1626 1641 1314
mean value [µg/L] 6.7 825 1163 1626 1639 1312
Day 7
Water Replicate 1 [µg/L] 32.8 56.3 46.8 54.5 83.5 95.9
Replicate 2 [µg/L] 34.0 54.3 46.8 55.4 84.2 94.8
mean value [µg/L] 33.4 55.3 46.8 54.9 83.9 95.4
Day 14
Water Replicate 1 [µg/L] 51.4 58.2 61.7 60.9 134.6 101.6
Replicate 2 [µg/L] 52.6 61.5 59.2 61.4 135.8 102.1
mean value [µg/L] 52.0 59.8 60.4 61.2 135.2 101.9
Sample
Control 15 mg/L 24 mg/L 39 mg/L 63 mg/L 100 mg/L Ti [mg/L] 9.0 1 14.4 1 23.4 1 37.8 1 59.9 1 Day 28
Water Replicate 1 [µg/L] 2.82 1.83 0.979 0.266 1.35 3.49
Replicate 2 [µg/L] 3.31 1.88 0.982 0.089 2.59 2.77
MW [µg/L] 3.07 1.86 0.981 0.177 1.97 3.13
Sediment [mg/kg] 1084 1080 1163 1082 1161 1228
Sample -
control - [mg/kg] -- -4 79 -2 77 144
Recovery 2 [%] -- -- 203 -- 77 150
Sediment background +
addition [mg/kg] 1084 1099 1108 1123 1147 1184
Recovery [%] -- 98 105 96 101 104
1 nominal values for Ti (calculated from TiO2 concentrations); 2 Recovery considering the amount of sediment (370 g dry weight) in the vessel and assuming 100% of Ti in the sediment;
Due to the activity of the chironomids in the sediment, settled TiO2 nanoparticles were trans-ferred into the sediment. During the exposure period of 28 days the settled white powder of TiO2 on the surface of the sediment vanished. It is assumed that during their development the organisms were mainly exposed to TiO2 nanoparticles present in the sediment. Exposure via the water phase after hatching and until leaving the aqueous compartment is considered to be negligible.
Exposure
Summarised results are presented in Effects
Table 89 and Table 90.
No significant effect for the total midges, the males and females was observed.
The LOEC, EC10, EC20, and EC50 values of the biological endpoints were > 100 mg/L. The NOEC was ≥ 100 mg/L.
Neither physical nor pathological symptoms were obtained. All specimens gave the impres-sion of healthy condition.
Physical/pathological symptoms and changes in behaviour
The results of emergence are presented in Emergence rate
Table 89.
No concentration/effect dependency on the emergence rate due to NM-101 was detected.
The NOEC (no observed effect concentration) for the tested species Chironomus riparius was found to be > 100 mg/L for males, females, and the combined sexes.
Table 89: NM-101 - Test with chironomids: emergence.
Emergence: number of individuals; emergence rate: % of introduced larvae; concentrations given as nominal values
Control 15 mg/L 24 mg/L 39 mg/L 63 mg/L 100 mg/L
Emerged midges [Ind.]
74 69 75 60 61 74
Emergence rate midges [%]
92.5 86.25 93.75 75.0 76.25 92.5
Emerged midges [males]
36 39 38 21 37 37
Emerged midges [females]
38 30 37 39 24 37
The results of development time and rate are presented as mean values ( Development time and rate
Table 90). No in-fluence on emergence due to NM-101 was detected.
Table 90: NM-101 - Test with chironomids: development time [d] and rate [1/d].
Concentrations given as nominal values.
Control 15 mg/L 24 mg/L 39 mg/L 63 mg/L 100 mg/L
Development time midges
18.5 20.3 18.3 18.7 18.1 18.0
Development rate midges
0.055 0.050 0.056 0.055 0.057 0.057
Development time males
17.0 19.3 17.0 17.1 17.6 16.5
Development rate males
0.060 0.053 0.060 0.060 0.058 0.061
Development time females
19.6 21.3 19.2 19.2 18.9 19.2
Development rate females
0.051 0.047 0.052 0.052 0.053 0.052
12.7 Validity 12.7.1 P25
The test is considered of limited validity since:
• The mean emergence in the controls was 68% at test end. The minimum as stated in the guideline is 70%.
• The development time of most adults of C. riparius in the controls was between 16 and 23 days after their insertion into the test vessels. This is within the required range of 12 – 23 days. However, one further animal developed each time at days 25, 26 and 29.
All further criteria mentioned in the guidelines are fulfilled.
• At the end of the test the dissolved oxygen concentration was at least 60% of the air saturation level at the temperature used; the pH in the overlaying water was in a range of 6 – 9 in all test vessels.
• The water temperature differed by less than ± 1 °C between the vessels and was maintained within the temperature range of 20 ± 2 °C.
The test was not repeated as no difference between the controls and the treated samples was observed.
12.7.2 NM-101
The test is considered valid since:
• The mean emergence in the controls was 92.5% (corresponding to more than the minimum 70% mentioned in the guideline) at test end.
Furthermore:
• The development time of the adults of C. riparius in the controls was between 16 and 21 days after their insertion into the test vessels.
• At the end of the test the dissolved oxygen concentration was at least 60% of the air saturation level at the temperature used; the pH of the overlaying water was in a range from 6 – 9 in all test vessels.
• The water temperature differed not more than ± 1°C between the vessels and was maintained within the temperature range of 20 ± 2°C.
